Cell cycle polynucleotides, polypeptides and uses thereof

ABSTRACT

The invention provides isolated polynucleotides and their encoded proteins that are involved in cell cycle regulation. The invention further provides vectors, recombinant expression cassettes, host cells, transgenic plants, and antibody compositions. The present invention provides methods and compositions relating to altering cell cycle protein content and/or composition of plants.

This application claims priority to U.S. Ser. No. 60/119,857 filed Feb.12, 1999, which is CIP of U.S. Ser. No. 09/398,858 filed Sep. 20, 1999which was converted from U.S. Ser. No. 60/101,551 filed Sep. 23, 1998,and which is a CIP of U.S. Ser. No. 09/257,131 filed Feb. 25, 1999 thedisclosures of which are incorporated herein by reference.

TECHNICAL FIELD

The present invention relates generally to plant molecular biology. Morespecifically, it relates to nucleic acids and methods for modulatingtheir expression in plants.

BACKGROUND OF THE INVENTION

Cell division plays an important role during all phases of plantdevelopment. The continuation of organogenesis and growth responses to achanging environment requires precise spatial, temporal anddevelopmental regulation of cell division activity in meristems (and incells with the capability to form new meristems such as in lateral rootformation). Such control of cell division is also important in organsthemselves (i.e. separate from meristems per se), for example, in leafexpansion, secondary growth, and endoreduplication.

A complex network controls cell proliferation in eukaryotes. Regulatorypathways communicate environmental constraints, such as nutrientavailability, mitogenic signals such as growth factors or hormones, ordevelopmental cues such as the transition from vegetative toreproductive. Ultimately, these regulatory pathways control the timing,frequency (rate), plane and position of cell divisions.

The basic mechanism of cell cycle control is conserved among eukaryotes.A catalytic protein serine/threonine kinase and an activating cyclinsubunit control progress through the cell cycle. The protein kinase isgenerally referred to as a cyclin-dependent-kinase (CDK), whose activityis modulated by phosphorylation and dephosphorylation events and bytheir association with regulatory subunits, called cyclins. CDKs requireassociation with cyclins for activation, and the timing of activation islargely dependent upon cyclin expression. CDKs are a family ofserine/threonine protein kinases that regulate individual cell cycletransitions.

Eukaryote genomes typically encode multiple cyclin and CDK genes. Inhigher eukaryotes, different members of the CDK family act in differentstages of the cell cycle. Cyclin genes are classified according tosequence, the timing of their appearance or activity during the cellcycle, and the cell cycle regulatory proteins with which they interact.In addition to cyclin and CDK subunits, CDKs are often physicallyassociated with other proteins that alter localization, substratespecificity, or activity. A few examples of such CDK interactingproteins are the CDK inhibitors, members of theRetinoblastoma-associated protein (Rb) family, and the ConstitutiveKinase Subunit (CKS).

The protein kinase activity of the complex is regulated by feedbackcontrol at certain checkpoints. At such checkpoints the CDK activitybecomes limiting for further progress. When the feedback control networksenses the completion of a checkpoint, CDK is activated and the cellpasses through to the next checkpoint. Changes in CDK activity areregulated at multiple levels, including reversible phosphorylation ofthe cell cycle factors, changes in subcellular localization of thecomplex, and the rates of synthesis and destruction of limitingcomponents. P. W. Doerner, Cell Cycle Regulation in Plants, PlantPhysoil., 106:823-827 (1994).

Plants have unique developmental features that distinguish them fromother eukaryotes. Plant cells do not migrate, and thus only celldivision, expansion and programmed cell death determine morphogenesis.Organs are formed throughout the entire life span of the plant fromspecialized regions called meristems. In addition, many differentiatedcells have the potential to both dedifferentiate and to reenter the cellcycle. There are also numerous examples of plant cell types that undergoendoreduplication, a process involving nuclear multiplication withoutcytokinesis. The study of plant cell cycle control genes is expected tocontribute to the understanding of these unique phenomena. O. Shaul etal., Regulation of Cell Division in Arabidopsis, Critical Reviews inPlant Sciences, 15(2):97-112 (1996).

Cell division in higher eukaryotes is controlled by two main checkpointsin the cell cycle that prevent the cell from entering either M- orS-phase of the cycle prematurely. Evidence from yeast and mammaliansystems has shown that over-expression of key cell cycle activatinggenes can either trigger cell division in non-dividing cells, orstimulate division in previously dividing cells (i.e. the duration ofthe cell cycle is decreased and cell size is reduced). Examples of geneswhose over-expression has been shown to stimulate cell division includecyclins (see, e.g. Doerner et al., Nature (1996) 380:520-423; Gudas etal., Mol. Cell. Biol. (1999) 19:612-622; Wang et al., Nature (1994)369:669-671; Quelle et al., Genes Dev. (1993) 7:1559-1571, E2Ftranscription factors (see, e.g. Johnson et al., Nature (1993)365:349-352; Lukas et al., (1996) Mol. Cell. Biol. 16:1047-1057), cdc25(see, e.g. Bell et al., (1993) Plant Molecular Biology 23:445-451;Draetta et al., (1996) BBA 1332:53-63), and mdm2 (see, e.g. Teoh et al.,(1997) Blood 90:1982-1992). Conversely, other gene products have beenfound to participate in negative regulation and/or checkpoint control,effectively blocking or retarding progression through the cell cycle.(see MacLachlan et al., (1995) Critical Rev. Eukaroytic Gene Expression5(2):127-156).

Current methods for genetic engineering in agronomically important cropssuch as maize and soybean require a specific cell type as the recipientof new DNA. In maize, these cells are found in relativelyundifferentiated, rapidly growing callus cells or on the scutellarsurface of the immature embryo (which gives rise to callus).Irrespective of the delivery method currently used, DNA is introducedinto literally thousands of cells, yet transformants are recovered atfrequencies of 10⁻⁵ relative to transiently-expressing cells. Insoybean, these cells are found in relatively undifferentiated, rapidlygrowing callus or suspension cells, or in nodal meristematic regions ofthe plant. Exacerbating this problem, the trauma that accompanies DNAintroduction directs recipient cells into cell cycle arrest andaccumulating evidence suggests that many of these cells are directedinto apoptosis or programmed cell death. (Bowen et al., TucsonInternational Mol. Biol. Meetings). It would therefore be desirable toincrease transformation efficiency.

Over the period between 1950 and 1980, the increase in maize productionworldwide outpaced both wheat and rice. Despite a temporary downswing inthe early to mid-1980's (due to both environmental and politicalfactors) world maize production has risen steadily from around 145million tons in 1950 to nearly 500 million tons by 1990. Increases inyield and harvested area have been the predominant contributors toenhanced world production; with yield playing the major role inindustrialized countries and area expansion being most important indeveloping countries. Yet, over the next ten years it's also predictedthat meeting the demand for corn worldwide will require an additional20% over current production (Dowswell, C. R., Paliwal, R. L., Cantrell,R. P., 1996, Maize in the Third World, Westview Press, Boulder, Colo.).

The components most often associated with maize productivity are grainyield or whole-plant harvest for animal feed (in the forms of silage,fodder, or stover). Thus the relative growth of the vegetative orreproductive organs might be preferred, depending on the ultimate use ofthe crop. Whether the whole plant or the ear are harvested, overallyield will depend strongly on vigor and growth rate. In modern maizehybrids, the impact of heterosis on overall plant vigor and yield hasbeen unarguably demonstrated (Duvick, D. N.,1984, In: Geneticcontributions to yield gains in five major crop plants. W. R. Fehr, ed.CSSA, Madison, Wis.).

Corn breeders since the 1930's have been selectively breeding byidentifying inbreds that in combination produce hybrid vigor well beyondeither parent. Surprisingly little is known about why hybrids are somuch larger than their parent inbreds, although there are someinteresting observations in the literature. In metabolic studies,heterosis (increases over either parent) has been observed forphysiological traits such as P uptake by roots (Baliger and Barber,1979; Nielsen and Barber, 1978), but for many enzymatic traits thehybrid is often intermediate to the inbred parents (Hageman, R. H.,Leng, E. R., Dudley, J. W. 1967. Adv. Agron. 19:45-86; Chevalier, P.,Schrader, L. E. 1977. Crop Sci. 17:897-901; Schrader, L. E. 1974. CropSci. 14:201-205; Schrader, L. E. 1985. PP 79-89. In: Exploitation ofphysiological and genetic variability to enhance crop productivity.Harper, J. E. ed. Am. Soc. Plant Physiol. Rockville, Md., Schrader, L.E., Cataldo, D. A., Peterson, D. M., Vogelzang, R. D. 1974. PlantPhysiol. 32:337-341).

Anatomical data is less confusing. In summarizing data from an earlierpublication, Kiesselbach states that approximately 10% of the increasedvigor of the hybrid over its inbred parents is due to cell enlargement,and 90% can be accounted for simply by increased cell numbers(Kiesselbach, T. A. 1922, 1949. The Structure and Reproduction of Corn,Nebraska Agric. Exp. Stn. Res. Bull. 161). Recently it was shown thatoverexpressing a B cyclin in Arabidopsis resulted in increased rootbiomass and the root cells were smaller (indicative of accelerated celldivision), but the overall plant morphology was not perturbed (Doerneret al., 1996).

SUMMARY OF THE INVENTION

The invention provides isolated polynucleotides and their encodedproteins that are involved in cell cycle regulation. The inventionfurther provides vectors, recombinant expression cassettes, host cells,transgenic plants, and antibody compositions. The present inventionprovides methods and compositions relating to altering cell cycleprotein content and/or composition of plants.

Definitions

The term “isolated” refers to material, such as a nucleic acid or aprotein, which is: (1) substantially or essentially free from componentswhich normally accompany or interact with the material as found in itsnaturally occurring environment or (2) if the material is in its naturalenvironment, the material has been altered by deliberate humanintervention to a composition and/or placed at a locus in the cell otherthan the locus native to the material.

As used herein, “nucleic acid” means a polynucleotide and includessingle or double-stranded polymer of deoxyribonucleotide orribonucleotide bases. Nucleic acids may also include modifiednucleotides that permit correct read through by a polymerase and do notalter the expression of a polypeptide encoded by the polynucleotide.

As used herein, “CycE polynucleotide” means a polynucleotide whichencodes a polypeptide that i) binds to Cdk2 and Rb proteins, ii)contains a cyclin box (Jeffrey et al. 1995, Nature 367:313-320P, andiii) contains the conserved motif TTPXS near the carboxy-terminus.

As used herein, “polypeptide” means proteins, protein fragments,modified proteins, amino acid sequences and synthetic amino acidsequences. The polypeptide may be glycosylated or not.

As used herein, “plant” includes but is not limited to plant cells,plant tissue and plant seeds.

By “fragment” is intended a portion of the nucleotide sequence or aportion of the amino acid sequence and hence protein encoded thereby.Preferably fragments of a nucleotide sequence may encode proteinfragments that retain the biological activity of the native nucleicacid. However, fragments of a nucleotide sequence which are useful ashybridization probes generally do not encode fragment proteins retainingbiological activity. Fragments of a nucleotide sequence are generallygreater than 10 nucleotides, preferably at least 20 nucleotides and upto the entire nucleotide sequence encoding the proteins of theinvention. Generally probes are less than 1000 nucleotides andpreferably less than 500 nucleotides. Fragments of the invention includeantisense sequences used to decrease expression of the inventive nucleicacids. Such antisense fragments may vary in length ranging from at leastabout 20 nucleotides, about 50 nucleotides, about 100 nucleotides, up toand including the entire coding sequence.

By “variants” is intended substantially similar sequences. Generally,nucleic acid sequence variants of the invention will have at least 50%,55%, 60, 65%, 70%, 75%, 80%, 85%, or preferably 90%, more preferably atleast 95% and most preferably at least 98% sequence identity to thenative nucleotide sequence. Generally, polypeptide sequence variants ofthe invention will have at least about 50%, 55%, 60%, 65%, 70%, 75% 80%,85%, 90%, 95% or at least 98% sequence identity to the native protein.

As used herein, “sequence identity” in the context of two nucleic acidsequences includes reference to the residues in the two sequences thatare the same when aligned for maximum correspondence over the entirecoding sequence of the present polynucleotides. As used herein, sequenceidentity is determined using the GCG/bestfit program, GAP 10 using a gapcreation penalty of 50 and a gap extension penalty of 3.

GAP uses the algorithm of Needleman and Wunsch (J. Mol. Biol.48:443-453, 1970) to find the alignment of two complete sequences thatmaximizes the number of matches and minimizes the number of gaps.Default gap creation penalty values and gap extension penalty values inVersion 10 of the Wisconsin Genetics Software Package for proteinsequences are 8 and 2, respectively. For nucleotide sequences thedefault gap creation penalty is 50 while the default gap extensionpenalty is 3. The gap creation and gap extension penalties can beexpressed as an integer selected from the group of integers consistingof from 0 to 200. Thus, for example, the gap creation and gap extensionpenalties can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65 or greater. The scoring matrix used in Version 10of the Wisconsin Genetics Software Package is BLOSUM62 (see Henikoff &Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915).

As used herein, “sequence similarity” or “sequence identity” in thecontext of two polypeptide sequences includes reference to the residuesin the two sequences that are the same when aligned for maximumcorrespondence over the entire sequence of the present polypeptides. Asused herein, sequence similarity is determined using the GCG/bestfitprogram, GAP 10 using a gap creation penalty of 8 and a gap extensionpenalty of 2.

Other methods of alignment of sequences for comparison are well-known inthe art. Optimal alignment of sequences for comparison may be conductedby the local homology algorithm of Smith and Waterman, Adv. Appl. Math.2:482 (1981); by the homology alignment algorithm of Needleman andWunsch, J. Mol. Biol. 48:443 (1970); by the search for similarity methodof Pearson and Lipman, Proc. Natl. Acad. Sci. 85:2444 (1988); bycomputerized implementations of these algorithms, including, but notlimited to: CLUSTAL in the PC/Gene program by Intelligenetics, MountainView, Calif.; GAP, BESTFIT, BLAST, FASTA, and TFASTA in the WisconsinGenetics Software Package, Genetics Computer Group (GCG), 575 ScienceDr., Madison, Wis., USA; the CLUSTAL program is well described byHiggins and Sharp, Gene 73:237-244 (1988); Higgins and Sharp, CABIOS5:151-153 (1989); Corpet et al., Nucleic Acids Research 16:10881-90(1988); Huang et al., Computer Applications in the Biosciences 8:155-65(1992), and Pearson et al., Methods in Molecular Biology 24:307-331(1994).

The BLAST family of programs which can be used for database similaritysearches includes: BLASTN for nucleotide query sequences againstnucleotide database sequences; BLASTX for nucleotide query sequencesagainst protein database sequences; BLASTP for protein query sequencesagainst protein database sequences; TBLASTN for protein query sequencesagainst nucleotide database sequences; and TBLASTX for nucleotide querysequences against nucleotide database sequences. See Current Protocolsin Molecular Biology, Chapter 19, Ausubel et al., Eds., GreenePublishing and Wiley-Interscience, New York (1995). Software forperforming BLAST analyses is publicly available, e.g., through theNational Center for Biotechnology Information(http://www.ncbi.nlm.nih.gov/). The BLAST algorithm performs astatistical analysis of the similarity between two sequences (see, e.g.,Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5877 (1993)). Onemeasure of similarity provided by the BLAST algorithm is the smallestsum probability (P(N)), which provides an indication of the probabilityby which a match between two nucleotide or amino acid sequences wouldoccur by chance.

By “functionally equivalent” is intended that the sequence of thevariant defines a chain that produces a protein having substantially thesame biological effect as the native protein of interest. The variant iscatalytically active.

By “modulate” is intended to increase, decrease, influence or change.

DETAILED DESCRIPTION OF THE INVENTION

As part of a complex with CDK2, Cyclin E (CycE) protein is an integralcomponent required for phosphorylation of retinoblastoma. Thephosphorylation of Rb results in the release of E2F, which thenactivates transcription of numerous genes involved in DNA replication.Thus CycE plays a significant role in the transition from G1 to S phaseof the cell cycle. Similar to Cyclin-D (another G1-S phase stimulatingprotein) CycE genes from heterologous species have been found tocomplement Saccharomyces cerevisiae cells lacking the G1 cyclin functionrequired for progression through START. CycE overexpression has beenfound to stimulate S-phase in various cell types in both Drosophila andmammalian cells (Ohtsubo, M., Roberts, J. M., 1993, Science259:1908-1912; Wimmels, A., Lucibello, F. C., Sewing, A., Adolf, S,Muller, R., 994, Oncogene 9:995-997; Resnitzky, D. M. G., Bujard, H.,Reed, S. I., 1994, Mol Cell Biol. 14:1669-1679; Ohtsubo, M., Theadoras,A. M., Schumacher, J., Roberts, J. M., Pagano, M., 1995, Mol Cell Biol.15:2612-2624. Evidence across a variety of fauna including Homo sapiens,Drosophila melanogaster, Xenopus laevis, zebrafish and mice suggeststhat the role of CycE is similar across these genera; activity of thisprotein promotes cell cycle entry into S-phase and is involved in suchprocesses as endocycling and organ pattern development.

Cells transformed to modulate the level of polypeptides that stimulatethe transition of G1 to S phase will increase transformation frequenciescompared to non-transformed plants. The transformation can be transientor stable, thus DNA, RNA, or proteins can be introduced into the cells.Proteins that influence the transition from the G1 to S phase includeCycD, CycE, E2F, RepA, cdk2, cdk4, Rb, or CKI. If the cell istransformed with DNA, the DNA is operably linked to a promoter. In orderto stimulate transition from the G1 to S phase levels of CycD, CycE,E2F, Geminiviral replication protein such as RepA, cdk2, or cdk4 proteinare increased, levels of Rb or CKI are decreased.

The above polypeptides or polynucleotides can be introduced into hostcells by known methods to enhance transformation efficiency. Sequencesfrom various sources are known. For example Wheat Dwarf Virus Rep andRepA sequences are in GenBank Accession No. X82104 and MSV C1 AccessionNo. AJ012641; Tomato Golden Mosaic Virus replication proteins A11, A12,and A13 in GenBank Accession No. K02029 and Embo J. 3, 2197-2205 (1984)Hamilton, W. D. O. et al.; Beet Curly Top Virus replication protein inGenBank Accession No. X97203 and Dur. J. Plant Pathol., 104, 77-84(1999) Briddon, R. W.; cdk2 Est from soybean in GenBank Accession No.AW279429; Homo sapiens cdk2 in GenBank Accession No. NM 001798 andNature 353 (6340), 1174-177 (1991) Tsai, L. H. et al.; cdk4 in soybeanin GenBank Accession No. AW 164283; Homo sapiens cdk4 in GenBankAccession No. NM 000075 and Cytogenet. Cell Genet 66(1), 72-74 (1994)Demetrick et al.; Chromosome Res. 3 (4), 261-262 (1995) Mitchel et al.;Nature Genet. 12 (1), 97-99 (1996) Zuo, L.; rice cdc2 in GenBankAccession No. X60375 and Mol. Gen. Genet. 233 (1-2), 10-16 (1992),Hashimoto et al.; maize cdc2 in GenBank Accession No. M60526 and Proc.Natl. Acad. Sci. U.S.A. 88, 3377-3381 (1991) Colassanti et al.; Homosapiens cdk7 in GenBank Accession No. NM 001799 and Oncogene 9(11),3127-3138 (1998) Darbon et al.; tobacco CycD in GenBank Accession No.AJ011894, AJ011893, AJ011892, and Plant Physiol. 119, 343-351 (1999)Murray, J. A. H.; pea CycD in GenBank Accession No. AB008188 and PlantCell Physiol. 39 (3), 255-262 (1998) Shimizu, S. and Mori, H.;Arabidopsis CycD in GenBank Accession No. X83369, X83370 and X83371 andPlant Cell 7 (1), 85-103 (1995) Murray, J. A. H.; C. rubrum CycD inGenBank Accession No. Y10162 Renz et al.; human CycE in GenBankAccession No. L48996 and Proc. Natl. Acad. Sci. U.S.A. 92 (26),12146-12150 (1995) Ohtani et al.; D. melanogaster CycE type 1 in GenBankAccession No. X75026 and X75027 and Development 119 (3), 673-690 (1993)Richardson H. E. et al; wheat E2F in GenBank Accession No. AJ238590 andNucleic Acids Res. 27, 3427-3533 (1999) Ramirez-Parra, E.; tobacco E2Fin GenBank Accession No. AB025347 and FEBS Lett. 460, 117-122 (1999)Sekine, M.; Rb in GenBank Accession No. A68394 and WO 9747647 GutierrezA. C.; RRB2b and RRB2ba in GenBank Accession No. AF007795 and Mol. Cell.Biol. 17 (9), 5077-5086 (1997) Ach, R. A. et al; Zea mays Rb1 in GenBankAccession No. X98923 and Embo J. 15 (18), 4900-4908 (1996) Xie, Q. etal; ZmRb in GenBank Accession No. U52099 Grafi, G. et al; ArabidopsisCKI in WO 99/14331, U.S. Ser. No. 60/119,857 filed Feb. 12, 1999; U.S.Ser. No. 09/398,858 filed Sep. 20, 1999, U.S. Ser. No. 60/119,857 filedFeb. 12, 1999; and U.S. Ser. No. 09/257,131 filed Feb. 25, 1999 thedisclosures of which are incorporated herein by reference.

Because CycE can stimulate progression of cells into S phase, increasingCycE activity may be useful in terms of increasing integrationfrequencies during the transformation process. Stimulation of the G1/Stransition results in increased cell division in certain cases, and inthis regard, use of CycE to stimulate cell division may stimulate callusgrowth and/or growth in the whole plant (or in specific tissues wherethis activity is targeted).

We have successfully used the maize Cyclin D (CycD) gene fortransformation improvement. In GS3, transformation frequency was foundto improve by 2 to 3-fold when a ZmCycD gene was used. In order toobtain even higher transformation frequency and/or genotype independenttransformation improvement, identification and manipulation of suchfactors is useful.

The Rb/E2F pathway is a key control mechanism for G1/S progression inmost eukaryotic cells. Cyclin D is a key positive regulator of the G1/Stransition, bringing CDK4/6 to the vicinity of Rb/E2F and initiating thephosphorylation of Rb. Cyclin E continues this process by recruitingCDK2 to form an active complex, which completes the phosphorylation ofRb. Phosphorylation of Rb protein is necessary to release E2F for G1/Stransition. Recent evidence suggests that CycD/CDK4 or 6 mainly inhibitRb-HDAC interaction (interactions between Rb and histone deacetylases)whereas CycE/CDK2 directly inhibits Rb-E2F interaction.

Rb represses S-phase entry through two mechanisms: i) binding to andinactivating E2F, and ii) recruiting HDAC to participate in chromatinremodeling. Both E2F and HDAC bind to the A-B pocket of Rb. Disruptionof the A-B pocket leads to an inactive Rb. The C-domain in Rb providesdocking sites for CycD and CycE. Initial phosphorylation of the C-domainby CyD/CDK4 or 6 leads to an intramolecular binding of the C-domain tothe pocket, specifically, to the lysine patch surrounding the LXCXEbinding site in domain B. This intramolecular interaction inhibits thebinding of HDAC to the pocket and promotes the access of CycE/CDK2 tophospho-acceptor sites in the B-domain. Progressive phosphorylation ofthese B-domain phosphorylation sites by CycE/CDK2 completes thehyperphosphorylation of Rb. More importantly, phosphorylation of S-567by CycE/CDK2 leads to disruption of the A-B pocket, inhibition of theinteraction between Rb and E2F, and thus to a stimulation of the G1/Stransition.

Therefore, the CycE nucleic acid is a key positive regulator for S-phaseentry. Manipulation of plant CycE nucleic acids will improvetransformation, especially when used together with the Cyclin D gene.CycE expression stimulates the G1-S phase transition, and will thusincrease integration frequencies upon introduction of DNA into thesecells. Expression of CycE will also provide a positive growth advantagein transgenic cells (relative to non-transformed tissues), thusproviding a method for positive selection of transformants based ondifferential growth rates.

CycE appears to be an important component in the endoreduplicationprocess in Drosophila. Appropriately enhanced CycE overexpression maystimulate the endoreduplication process in maize, and could be used topurposefully stimulate endoreduplication in tissues where this processnormally does not occur, or to enhance this process in cells and/ortissues that normally undergo endoreduplication.

CycE may increase crop yield, growth and biomass accumulation. CycEexpression could stimulate cell division in specific tissues (undercontrol of a promoter specific to said tissue), increasing the relativegrowth of the targeted tissue (i.e. increased vegetative growth in thestem and/or leaves, increased ear size, kernel size, etc). The sequencecould also be used to block division in certain cells (i.e. as asterility method) using the CycE sequence in such well-known methods asantisense expression, co-suppression or hairpin technology to silenceendogenous CycE expression.

Other more specialized applications exist for these genes at the wholeplant level. It has been demonstrated that endoreduplication occurs innumerous cell types within plants, but this is particularly prevalent inmaize endosperm, the primary seed storage tissue. Under the direction ofendosperm-specific promoters, expression of CycE genes (and possiblyexpression of CycE in conjunction with genes that inhibit mitosis) willfurther stimulate the process of endoreduplication.

In addition to the positive influence of transient cell cyclestimulation, stable expression of positive cell cycle regulators wouldbe a benefit for positive selection schemes in the recovery oftransgenic plants and plant cells. In a population of cells and/orcallus growing in vitro, cells expressing a gene such as CycE will havea differential growth advantage based simply on their accelerateddivision rate. It would be expected that these transgenic cells orcell/clusters would grow more rapidly than their non-transformedcounterparts in culture, permitting ready identification oftransformants.

Such a positive growth advantage (imparted by expression of a gene suchas CycE, or CycE plus another cell cycle component), would also bebeneficial in other types of transformation strategies, including asexamples, protoplast transformation, leaf base transformation andtransformation of cells in meristems. Such growth stimulation may alsoextend transformation protocols to tissues normally no amenable toculture. Examples would include such tissues as portions of leaves (inwhich the cells do not normally divide), scutellum from recalcitrantinbreds (in which cells typically are not induced to divide in culture),cambial tissues, and nodal tissues, etc.

Of particular interest is the use of cell cycle genes such as CycE toimpart a positive growth advantage to cells in the meristem, includingapical initials. The apical initials in angiosperm shoot meristems aredefined by their position within the meristem. If an apical initial cellbecomes compromised relative to neighboring cells in the meristem, itwill be replaced by an adjacent neighbor that is not at a disadvantage.This new cell assumes the role of the apical initial. Conversely,transgenic cells adjacent to the apical initials with a positive growthadvantage can, over time (i.e. through successive cell generations),out-compete the wild-type apical initials, eventually replacing thesecells and establishing a homogeneous transformed meristem. There canalso be organ and/or whole plant impacts to such cell cycle transgeneexpression.

References

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Geng Y, W Whoriskey, M Y Park, R T Bronson, R H Medema, T Li, R AWeinberg, and P Sicinski 1999, Rescue of Cyclin D1 deficiency byknocking Cyclin E. Cell 97:767-777.

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Nucleic Acids

The isolated nucleic acids of the present invention can be made using(a) standard recombinant methods, (b) synthetic techniques, orcombinations thereof. In some embodiments, the polynucleotides of thepresent invention will be cloned, amplified, or otherwise constructedfrom a monocot or dicot. In preferred embodiments the monocot is corn,sorghum, barley, wheat, millet, or rice. Preferred dicots includesoybeans, sunflower, safflower, canola, alfalfa, cotton, potato, orcassava.

Functional fragments included in the invention can be obtained usingprimers that selectively hybridize under stringent conditions. Primersare generally at least 12 bases in length and can be as high as 200bases, but will generally be from 15 to 75, preferably from 15 to 50.Functional fragments can be identified using a variety of techniquessuch as restriction analysis, Southern analysis, primer extensionanalysis, and DNA sequence analysis.

The present invention includes a plurality of polynucleotides thatencode for the identical amino acid sequence. The degeneracy of thegenetic code allows for such “silent variations” which can be used, forexample, to selectively hybridize and detect allelic variants ofpolynucleotides of the present invention. Additionally, the presentinvention includes isolated nucleic acids comprising allelic variants.The term “allele” as used herein refers to a related nucleic acid of thesame gene.

Variants of nucleic acids included in the invention can be obtained, forexample, by oligonucleotide-directed mutagenesis, linker-scanningmutagenesis, mutagenesis using the polymerase chain reaction, and thelike. See, for example, Ausubel, pages 8.0.3-8.5.9. Also, see generally,McPherson (ed.), DIRECTED MUTAGENESIS: A Practical approach, (IRL Press,1991). Thus, the present invention also encompasses DNA moleculescomprising nucleotide sequences that have substantial sequencesimilarity with the inventive sequences.

Variants included in the invention may contain individual substitutions,deletions or additions to the nucleic acid or polypeptide sequences.Such changes will alter, add or delete a single amino acid or a smallpercentage of amino acids in the encoded sequence. Variants are referredto as “conservatively modified variants” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.When the nucleic acid is prepared or altered synthetically, advantagecan be taken of known codon preferences of the intended host.

The present invention also includes “shufflents” produced by sequenceshuffling of the inventive polynucleotides to obtain a desiredcharacteristic. Sequence shuffling is described in PCT publication No.96/19256. See also, Zhang, J.-H., et al. Proc. Natl. Acad. Sci. USA94:4504-4509 (1997).

The present invention also includes the use of 5′ and/or 3′ UTR regionsfor modulation of translation of heterologous coding sequences. Positivesequence motifs include translational initiation consensus sequences(Kozak, Nucleic Acids Res. 15: 8125 (1987)) and the 7-methylguanosinecap structure (Drummond et al., Nucleic Acids Res. 13:7375 (1985)).Negative elements include stable intramolecular 5′ UTR stem-loopstructures (Muesing et al., Cell 48:691 (1987)) and AUG sequences orshort open reading frames preceded by an appropriate AUG in the 5′ UTR(Kozak, supra, Rao et al., Mol. and Cell. Biol. 8:284 (1988)).

Further, the polypeptide-encoding segments of the polynucleotides of thepresent invention can be modified to alter codon usage. Altered codonusage can be employed to alter translational efficiency and/or tooptimize the coding sequence for expression in a desired host or tooptimize the codon usage in a heterologous sequence for expression inmaize. Codon usage in the coding regions of the polynucleotides of thepresent invention can be analyzed statistically using commerciallyavailable software packages such as “Codon Preference” available fromthe University of Wisconsin Genetics Computer Group (see Devereaux etal., Nucleic Acids Res. 12:387-395 (1984)) or MacVector 4.1 (EastmanKodak Co., New Haven, Conn.).

For example, the inventive nucleic acids can be optimized for enhancedexpression in organisms of interest. See, for example, EPA0359472;WO91/16432; Perlak et al. (1991) Proc. Natl. Acad. Sci. USA88:3324-3328; and Murray et al. (1989) Nucleic Acids Res. 17:477-498. Inthis manner, the genes can be synthesized utilizing species-preferredcodons. See, for example, Murray et al. (1989) Nucleic Acids Res.17:477-498, the disclosure of which is incorporated herein by reference.

The present invention provides subsequences comprising isolated nucleicacids containing at least 20 contiguous bases of the inventivesequences. For example the isolated nucleic acid includes thosecomprising at least 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, or 100or more contiguous nucleotides of the inventive sequences. Subsequencesof the isolated nucleic acid can be used to modulate or detect geneexpression by introducing into the subsequences compounds which bind,intercalate, cleave and/or crosslink to nucleic acids.

The nucleic acids of the invention may conveniently comprise amulti-cloning site comprising one or more endonuclease restriction sitesinserted into the nucleic acid to aid in isolation of thepolynucleotide. Also, translatable sequences may be inserted to aid inthe isolation of the translated polynucleotide of the present invention.For example, a hexa-histidine marker sequence provides a convenientmeans to purify the proteins of the present invention.

A polynucleotide of the present invention can be attached to a vector,adapter, promoter, transit peptide or linker for cloning and/orexpression of a polynucleotide of the present invention. Additionalsequences may be added to such cloning and/or expression sequences tooptimize their function in cloning and/or expression, to aid inisolation of the polynucleotide, or to improve the introduction of thepolynucleotide into a cell. Use of cloning vectors, expression vectors,adapters, and linkers is well known and extensively described in theart. For a description of such nucleic acids see, for example,Stratagene Cloning Systems, Catalogs 1995, 1996, 1997 (La Jolla,Calif.); and, Amersham Life Sciences, Inc, Catalog '97 (ArlingtonHeights, Ill.).

The isolated nucleic acid compositions of this invention, such as RNA,cDNA, genomic DNA, or a hybrid thereof, can be obtained from plantbiological sources using any number of cloning methodologies known tothose of skill in the art. In some embodiments, oligonucleotide probesthat selectively hybridize, under stringent conditions, to thepolynucleotides of the present invention are used to identify thedesired sequence in a cDNA or genomic DNA library.

Exemplary total RNA and mRNA isolation protocols are described in PlantMolecular Biology: A Laboratory Manual, Clark, Ed., Springer-Verlag,Berlin (1997); and, Current Protocols in Molecular Biology, Ausubel, etal., Eds., Greene Publishing and Wiley-lnterscience, New York (1995).Total RNA and mRNA isolation kits are commercially available fromvendors such as Stratagene (La Jolla, Calif.), Clonetech (Palo Alto,Calif.), Pharmacia (Piscataway, N.J.), and 5′-3′ (Paoli, Pa.). See also,U.S. Pat. Nos. 5,614,391;and, 5,459,253.

Typical cDNA synthesis protocols are well known to the skilled artisanand are described in such standard references as: Plant MolecularBiology: A Laboratory Manual, Clark, Ed., Springer-Verlag, Berlin(1997); and, Current Protocols in Molecular Biology, Ausubel, et al.,Eds., Greene Publishing and Wiley-lnterscience, New York (1995). cDNAsynthesis kits are available from a variety of commercial vendors suchas Stratagene or Pharmacia.

An exemplary method of constructing a greater than 95% pure full-lengthcDNA library is described by Carninci et al., Genomics, 37:327-336(1996). Other methods for producing full-length libraries are known inthe art. See, e.g., Edery et al., Mol. Cell Biol., 15(6):3363-3371(1995); and, PCT Application WO 96/34981.

It is often convenient to normalize a cDNA library to create a libraryin which each clone is more equally represented. A number of approachesto normalize cDNA libraries are known in the art. Construction ofnormalized libraries is described in Ko, Nucl. Acids. Res.,18(19):5705-5711 (1990); Patanjali et al., Proc. Natl. Acad. U.S.A.,88:1943-1947 (1991); U.S. Pat. Nos. 5,482,685 and 5,637,685; and Soareset al., Proc. Natl. Acad. Sci. USA, 91:9228-9232 (1994).

Subtracted cDNA libraries are another means to increase the proportionof less abundant cDNA species. See, Foote et al. in, Plant MolecularBiology: A Laboratory Manual, Clark, Ed., Springer-Verlag, Berlin(1997); Kho and Zarbl, Technique, 3(2):58-63 (1991); Sive and St. John,Nucl. Acids Res., 16(22):10937 (1988); Current Protocols in MolecularBiology, Ausubel, et al., Eds., Greene Publishing andWiley-lnterscience, New York (1995); and, Swaroop et al., Nucl. AcidsRes., 19(8):1954 (1991). cDNA subtraction kits are commerciallyavailable. See, e.g., PCR-Select (Clontech).

To construct genomic libraries, large segments of genomic DNA aregenerated by random fragmentation. Examples of appropriate molecularbiological techniques and instructions are found in Sambrook, et al.,Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring HarborLaboratory Vols. 1-3 (1989), Methods in Enzymology, Vol. 152: Guide toMolecular Cloning Techniques, Berger and Kimmel, Eds., San Diego:Academic Press, Inc. (1987), Current Protocols in Molecular Biology,Ausubel, et al., Eds., Greene Publishing and Wiley-lnterscience, NewYork (1995); Plant Molecular Biology: A Laboratory Manual, Clark, Ed.,Springer-Verlag, Berlin (1997). Kits for construction of genomiclibraries are also commercially available.

The cDNA or genomic library can be screened using a probe based upon thesequence of a nucleic acid of the present invention such as thosedisclosed herein. Probes may be used to hybridize with genomic DNA orcDNA sequences to isolate homologous genes in the same or differentplant species. Those of skill in the art will appreciate that variousdegrees of stringency of hybridization can be employed in the assay; andeither the hybridization or the wash medium can be stringent. The degreeof stringency can be controlled by temperature, ionic strength, pH andthe presence of a partially denaturing solvent such as formamide.

Typically, stringent hybridization conditions will be those in which thesalt concentration is less than about 1.5 M Na ion, typically about 0.01to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide.

For purposes of defining the invention, the hybridization is preferablyconducted under low stringency conditions which include hybridizationwith a buffer solution of 30% formamide, 1 M NaCl, 1% SDS (sodiumdodecyl sulfate) at 37° C., and a wash in 1× to 2×SSC (20×SSC=3.0 MNaCl/0.3 M trisodium citrate) at 50° C. More preferably thehybridization is conducted under moderate stringency conditions whichinclude hybridization in 40% formamide, 1 M NaCl, 1% SDS at 37° C., anda wash in 0.5× to 1×SSC at 55° C. Most preferably the hybridization isconducted under high stringency conditions which include hybridizationin 50% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.1×SSC at60° C. The time for conducting the hybridization is not critical and isgenerally in the range of from 4 to 16 hours.

An extensive guide to the hybridization of nucleic acids is found inTijssen, Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2“Overview of principles of hybridization and the strategy of nucleicacid probe assays”, Elsevier, N.Y. (1993); and Current Protocols inMolecular Biology, Chapter 2, Ausubel, et al., Eds., Greene Publishingand Wiley-lnterscience, New York (1995). Often, cDNA libraries will benormalized to increase the representation of relatively rare cDNAs.

The nucleic acids of the invention can be amplified from plant nucleicacid samples using amplification techniques. For instance, polymerasechain reaction (PCR) technology can be used to amplify the sequences ofpolynucleotides of the present invention and related genes directly fromgenomic DNA or cDNA libraries. PCR and other in vitro amplificationmethods may also be useful, for example, to clone nucleic acid sequencesthat code for proteins to be expressed, to make nucleic acids to use asprobes for detecting the presence of the desired mRNA in samples, fornucleic acid sequencing, or for other purposes.

The nucleic acid library can be a cDNA library, a genomic library, or alibrary generally constructed from nuclear transcripts at any stage ofintron processing. Libraries can be made from a variety of planttissues. Good results have been obtained using mitotically activetissues such as shoot meristems, shoot meristem cultures, embryos,callus and suspension cultures, immature ears and tassels, and youngseedlings. The cDNA of the present invention was obtained fromdeveloping maize endosperm. Since cell cycle proteins are typicallyexpressed at specific cell cycle stages it may be possible to enrich forsuch rare messages using exemplary cell cycle inhibitors such asaphidicolin, hydroxyurea, mimosine, and double-phosphate starvationmethods to block cells at the G1/S boundary. Cells can also be blockedat this stage using the double phosphate starvation method. Hormonetreatments that stimulate cell division, for example cytokinin, wouldalso increase expression of the cell cycle RNA.

Examples of techniques useful for in vitro amplification methods arefound in Berger, Sambrook, and Ausubel, as well as Mullis et al., U.S.Pat. No. 4,683,202 (1987); and, PCR Protocols A Guide to Methods andApplications, Innis et al., Eds., Academic Press Inc., San Diego, Calif.(1990). Commercially available kits for genomic PCR amplification areknown in the art. See, e.g., Advantage-GC Genomic PCR Kit (Clontech).The T4 gene 32 protein (Boehringer Mannheim) can be used to improveyield of long PCR products.

PCR-based screening methods have also been described. Wilfinger et al.describe a PCR-based method in which the longest cDNA is identified inthe first step so that incomplete clones can be eliminated from study.BioTechniques, 22(3):481-486 (1997).

The sequences of the invention can be used to isolate correspondingsequences in other organisms, particularly other plants, moreparticularly, other monocots. In this manner, methods such as PCR,hybridization, and the like can be used to identify such sequenceshaving substantial sequence similarity to the sequences of theinvention. See, for example, Sambrook et al. (1989) Molecular Cloning: ALaboratory Manual (2d ed., Cold Spring Harbor Laboratory Press,Plainview, N.Y.), and Innis et al. (1990), PCR Protocols: A Guide toMethods and Applications (Academic Press, New York). Coding sequencesisolated based on their sequence identity to the entire inventive codingsequences set forth herein or to fragments thereof are encompassed bythe present invention.

The isolated nucleic acids of the present invention can also be preparedby direct chemical synthesis by methods such as the phosphotriestermethod of Narang et al., Meth. Enzymol. 68:90-99 (1979); thephosphodiester method of Brown et al., Meth. Enzymol. 68:109-151 (1979);the diethylphosphoramidite method of Beaucage et al., Tetra. Lett.22:1859-1862 (1981); the solid phase phosphoramidite triester methoddescribed by Beaucage and Caruthers, Tetra. Letts. 22(20):1859-1862(1981), e.g., using an automated synthesizer, e.g., as described inNeedham-VanDevanter et al., Nucleic Acids Res., 12: 6159-6168 (1984);and, the solid support method of U.S. Pat. No. 4,458,066. Chemicalsynthesis generally produces a single stranded oligonucleotide. This maybe converted into double stranded DNA by hybridization with acomplementary sequence, or by polymerization with a DNA polymerase usingthe single strand as a template. One of skill will recognize that whilechemical synthesis of DNA is limited to sequences of about 100 bases,longer sequences may be obtained by the ligation of shorter sequences.

Expression Cassettes

The present invention also includes expression cassettes comprisingisolated nucleic acids of the present invention. An expression cassettewill typically comprise a polynucleotide of the present inventionoperably linked to transcriptional initiation regulatory sequences whichwill direct the transcription of the polynucleotide in the intended hostcell, such as tissues of a transformed plant. Plant expression vectorsmay also include selectable marker.

The construction of expression cassettes that can be employed inconjunction with the present invention is well known to those of skillin the art in light of the present disclosure. See, e.g., Sambrook etal.; Molecular Cloning: A Laboratory Manual; Cold Spring Harbor, N.Y.;(1989); Gelvin et al.; Plant Molecular Biology Manual; (1990); PlantBiotechnology: Commercial Prospects and Problems, eds. Prakash et al.;Oxford & IBH Publishing Co.; New Delhi, India; (1993); and Heslot et al;Molecular Biology and Genetic Engineering of Yeasts; CRC Press, Inc.,USA; (1992); each incorporated herein in its entirety by reference.

Suitable promoter regulatory regions generally include a transcriptioninitiation start site, a ribosome-binding site, an RNA processingsignal, a transcription termination site, and/or a polyadenylationsignal. Useful promoters can confer inducible, constitutive,environmentally- or developmentally-regulated, or cell- ortissue-preferred/selective expression.

Examples of constitutive promoters include the cauliflower mosaic virus(CaMV) 35S transcription initiation region, the 1′- or 2′- promoterderived from T-DNA of Agrobacterium tumefaciens, the ubiquitin 1promoter, the Smas promoter, the cinnamyl alcohol dehydrogenase promoter(U.S. Pat. No. 5,683,439), the Nos promoter, the pEmu promoter, therubisco promoter, the GRP1-8 promoter and other transcription initiationregions from various plant genes known to those of skill.

Examples of inducible promoters are the Adh1 promoter that is inducibleby hypoxia or cold stress, the Hsp70 promoter which is inducible by heatstress, and the PPDK promoter which is inducible by light. Also usefulare promoters that are chemically inducible. Inducing expressionimmediately after DNA introduction will improve integration and promotea growth response caused by the induced gene. Inducing the gene at alater time will cause a differential growth response.

Examples of promoters under developmental control include promoters thatinitiate transcription preferentially in certain tissues, such asleaves, roots, fruit, seeds, or flowers. An anther specific promoter5126 is disclosed in (U.S. Pat. Nos. 5,689,049 and 5,689,051). Examplesof seed-preferred promoters include, but are not limited to, 27 kD gammazein promoter and waxy promoter, Boronat, A., Martinez, M. C., Reina,M., Puigdomenech, P. and Palau, J.; Isolation and sequencing of a 28 kDglutelin-2 gene from maize: Common elements in the 5′ flanking regionsamong zein and glutelin genes; Plant Sci. 47, 95-102 (1986) and Reina,M., Ponte, I., Guillen, P., Boronat, A. and Palau, J., Sequence analysisof a genomic clone encoding a Zc2 protein from Zea mays W64 A, NucleicAcids Res. 18(21), 6426 (1990). See the following site relating to thewaxy promoter: Kloesgen, R. B., Gierl, A., Schwarz-Sommer, Z S. andSaedler, H., Molecular analysis of the waxy locus of Zea mays, Mol. Gen.Genet. 203, 237-244 (1986). Promoters that express in the embryo,pericarp, and endosperm are disclosed in U.S. applications Ser. Nos.60/097,233 filed Aug. 20, 1998 and 60/098,230 filed Aug. 28, 1998. Thedisclosures each of these are incorporated herein by reference in theirentirety.

Either heterologous or non-heterologous (i.e., endogenous) promoters canbe employed to direct expression of the nucleic acids of the presentinvention. These promoters can also be used, for example, in expressioncassettes to drive expression of antisense nucleic acids to reduce,increase, or alter concentration and/or composition of the proteins ofthe present invention in a desired tissue.

If polypeptide expression is desired, it is generally desirable toinclude a polyadenylation region at the 3′-end of the polynucleotidecoding region. The polyadenylation region can be derived from thenatural gene, from a variety of other plant genes, or from T-DNA. The 3′end sequence to be added can be derived from, for example, the nopalinesynthase or octopine synthase genes, or alternatively from another plantgene, or less preferably from any other eukaryotic gene.

An intron sequence can be added to the 5′ untranslated region or thecoding sequence of the partial coding sequence to increase the amount ofthe mature message that accumulates. See for example Buchman and Berg,Mol. Cell Biol. 8:4395-4405 (1988); Callis et al., Genes Dev.1:1183-1200 (1987). Use of maize introns Adh1-S intron 1,2, and 6, theBronze-1 intron are known in the art. See generally, The Maize Handbook,Chapter 116, Freeling and Walbot, Eds., Springer, N.Y. (1994).

The vector comprising the sequences from a polynucleotide of the presentinvention will typically comprise a marker gene, which confers aselectable phenotype on plant cells. Usually, the selectable marker genewill encode antibiotic or herbicide resistance. Suitable genes includethose coding for resistance to the antibiotic spectinomycin orstreptomycin (e.g., the aada gene), the streptomycin phosphotransferase(SPT) gene coding for streptomycin resistance, the neomycinphosphotransferase (NPTII) gene encoding kanamycin or geneticinresistance, the hygromycin phosphotransferase (HPT) gene coding forhygromycin resistance.

Suitable genes coding for resistance to herbicides include those whichact to inhibit the action of acetolactate synthase (ALS), in particularthe sulfonylurea-type herbicides (e.g., the acetolactate synthase (ALS)gene containing mutations leading to such resistance in particular theS4 and/or Hra mutations), those which act to inhibit action of glutaminesynthase, such as phosphinothricin or basta (e.g., the bar gene), orother such genes known in the art. The bar gene encodes resistance tothe herbicide basta and the ALS gene encodes resistance to the herbicidechlorsulfuron.

Typical vectors useful for expression of nucleic acids in higher plantsare well known in the art and include vectors derived from thetumor-inducing (Ti) plasmid of Agrobacterium tumefaciens described byRogers et al., Meth. In Enzymol., 153:253-277 (1987). Exemplary A.tumefaciens vectors useful herein are plasmids pKYLX6 and pKYLX7 ofSchardl et al., Gene, 61:1-11 (1987) and Berger et al., Proc. Natl.Acad. Sci. U.S.A., 86:8402-8406 (1989). Another useful vector herein isplasmid pBI101.2 that is available from Clontech Laboratories, Inc.(Palo Alto, Calif.).

A variety of plant viruses that can be employed as vectors are known inthe art and include cauliflower mosaic virus (CaMV), geminivirus, bromemosaic virus, and tobacco mosaic virus.

A polynucleotide of the present invention can be expressed in eithersense or anti-sense orientation as desired. In plant cells, it has beenshown that antisense RNA inhibits gene expression by preventing theaccumulation of mRNA which encodes the enzyme of interest, see, e.g.,Sheehy et al., Proc. Nat'l. Acad. Sci. (USA) 85:8805-8809 (1988); andHiatt et al., U.S. Pat. No. 4,801,340.

Another method of suppression is sense suppression, or cosuppression.Introduction of nucleic acid configured in the sense orientation hasbeen shown to be an effective means by which to block the transcriptionof target genes. For an example of the use of this method to modulateexpression of endogenous genes see, Napoli et al., The Plant Cell2:279-289 (1990) and U.S. Pat. No. 5,034,323.

Gene expression can also be down-regulated by means of hairpintechnology, Waterhouse et al. Proc. Natl. Acad. Sci. USA 95 pp.1359-1364 (1998); Selker, Cell, Vol. 97, 157-160, Apr. 16, 1999; Grant,Cell, Vol. 96, 303-306, Feb. 5, 1999. Another method of down-regulationof the protein involves using PEST sequences that provide a target fordegradation of the protein.

Catalytic RNA molecules or ribozymes can also be used to inhibitexpression of plant genes. The inclusion of ribozyme sequences withinantisense RNAs confers RNA-cleaving activity upon them, therebyincreasing the activity of the constructs. The design and use of targetRNA-specific ribozymes is described in Haseloff et al., Nature334:585-591 (1988).

A variety of cross-linking agents, alkylating agents and radicalgenerating species as pendant groups on polynucleotides of the presentinvention can be used to bind, label, detect, and/or cleave nucleicacids. For example, Vlassov et al., Nucleic Acids Res (1986)14:4065-4076, describe covalent bonding of a single-stranded DNAfragment with alkylating derivatives of nucleotides complementary totarget sequences. A report of similar work by the same group is that byKnorre et al., Biochimie (1985) 67:785-789. Iverson and Dervan alsoshowed sequence-specific cleavage of single-stranded DNA mediated byincorporation of a modified nucleotide which was capable of activatingcleavage (J Am Chem Soc (1987) 109:1241-1243). Meyer et al., J Am ChemSoc (1989) 111:8517-8519, effect covalent crosslinking to a targetnucleotide using an alkylating agent complementary to thesingle-stranded target nucleotide sequence. A photoactivatedcrosslinking to single-stranded oligonucleotides mediated by psoralenwas disclosed by Lee et al., Biochemistry (1988) 27:3197-3203. Use ofcrosslinking in triple-helix forming probes was also disclosed by Homeet al., J Am Chem Soc (1990) 112:2435-2437. Use of N4, N4-ethanocytosineas an alkylating agent to crosslink to single-stranded oligonucleotideshas also been described by Webb and Matteucci, J Am Chem Soc (1986)108:2764-2765; Nucleic Acids Res (1986) 14:7661-7674; Feteritz et al.,J. Am. Chem. Soc. 113:4000 (1991). Various compounds to bind, detect,label, and/or cleave nucleic acids are known in the art. See, forexample, U.S. Pat. Nos. 5,543,507; 5,672,593; 5,484,908; 5,256,648; and,5,681941.

Proteins

Proteins of the present invention include proteins derived from thenative protein by deletion (so-called truncation), addition orsubstitution of one or more amino acids at one or more sites in thenative protein. Such variants may result from, for example, geneticpolymorphism or from human manipulation. Methods for such manipulationsare generally known in the art.

For example, amino acid sequence variants of the polypeptide can beprepared by mutations in the cloned DNA sequence encoding the nativeprotein of interest. Methods for mutagenesis and nucleotide sequencealterations are well known in the art. See, for example, Walker andGaastra, eds. (1983) Techniques in Molecular Biology (MacMillanPublishing Company, New York); Kunkel (1985) Proc. Natl. Acad. Sci. USA82:488-492; Kunkel et al. (1987) Methods Enzymol. 154:367-382; Sambrooket al. (1989) Molecular Cloning: A Laboratory Manual (Cold SpringHarbor, N.Y.); U.S. Pat. No. 4,873,192; and the references citedtherein; herein incorporated by reference. Guidance as to appropriateamino acid substitutions that do not affect biological activity of theprotein of interest may be found in the model of Dayhoff et al. (1978)Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found.,Washington, D.C.), herein incorporated by reference. Conservativesubstitutions, such as exchanging one amino acid with another havingsimilar properties, may be preferred.

If the enzyme activity is to be maintained, mutations made in the DNAencoding the variant protein should not place the sequence out ofreading frame and preferably will not create complementary regions thatcould produce secondary mRNA structure. See EP Patent ApplicationPublication No. 75,444.

The isolated proteins of the present invention include a polypeptidecomprising at least 23 contiguous amino acids encoded by any one of thenucleic acids of the present invention, or polypeptides that areconservatively modified variants thereof. The proteins of the presentinvention or variants thereof can comprise any number of contiguousamino acid residues from a polypeptide of the present invention, whereinthat number is selected from the group of integers consisting of from 23to the number of residues in a full-length polypeptide of the presentinvention. Optionally, this subsequence of contiguous amino acids is atleast 25, 30, 35, 40, 45 amino acids in length, often at least 50, 60,70, 80, or 90 amino acids in length.

The present invention includes catalytically active polypeptides (i.e.,enzymes). Catalytically active polypeptides will generally have aspecific activity of at least 20%, 30%, or 40%, and preferably at least50%, 60%, or 70%, and most preferably at least 80%, 90%, or 95% that ofthe native (non-synthetic), endogenous polypeptide. The invention alsoincludes polypeptides with much higher activity than the native protein.

Further, the substrate specificity (k_(cat)/K_(m)) is optionallysubstantially similar to the native (non-synthetic), endogenouspolypeptide. Typically, the K_(m) will be at least 30%, 40%, or 50%,that of the native (non-synthetic), endogenous polypeptide; and morepreferably at least 60%, 70%, 80%, or 90%. Methods of assaying andquantifying measures of enzymatic activity and substrate specificity(k_(cat)/K_(m)), are well known to those of skill in the art.

The present invention includes modifications that can be made to aninventive protein without diminishing its biological activity. Somemodifications may be made to facilitate the cloning, expression, orincorporation of the targeting molecule into a fusion protein. Suchmodifications are well known to those of skill in the art and include,for example, a methionine added at the amino terminus to provide aninitiation site, or additional amino acids (e.g., poly His) placed oneither terminus to create conveniently located restriction sites ortermination codons or purification sequences.

A protein of the present invention can be expressed in a recombinantlyengineered cell such as bacteria, yeast, insect, mammalian, orpreferably plant cells. The cells produce the protein in a non-naturalcondition (e.g., in quantity, composition, location, and/or time),because they have been genetically altered through human intervention todo so.

Typically, an intermediate host cell will be used in the practice ofthis invention to increase the copy number of the cloning vector. Withan increased copy number, the vector containing the nucleic acid ofinterest can be isolated in significant quantities for introduction intothe desired plant cells.

Host cells that can be used in the practice of this invention includeprokaryotes, including bacterial hosts such as Eschericia coli,Salmonella typhimurium, and Serratia marcescens. Eukaryotic hosts suchas yeast or filamentous fungi may also be used in this invention. Itpreferred to use plant promoters that do not cause expression of thepolypeptide in bacteria.

Commonly used prokaryotic control sequences include promoters such asthe beta lactamase (penicillinase) and lactose (lac) promoter systems(Chang et al., Nature 198:1056 (1977)), the tryptophan (trp) promotersystem (Goeddel et al., Nucleic Acids Res. 8:4057 (1980)) and the lambdaderived P L promoter and N-gene ribosome binding site (Shimatake et al.,Nature 292:128 (1981)). The inclusion of selection markers in DNAvectors transfected in E. coli is also useful. Examples of such markersinclude genes specifying resistance to ampicillin, tetracycline, orchloramphenicol.

The vector is selected to allow introduction into the appropriate hostcell. Bacterial vectors are typically of plasmid or phage origin.Expression systems for expressing a protein of the present invention areavailable using Bacillus sp. and Salmonella (Palva, et al., Gene22:229-235 (1983); Mosbach, et al., Nature 302: 543-545 (1983)).

Synthesis of heterologous proteins in yeast is well known. See Sherman,F., et al., Methods in Yeast Genetics, Cold Spring Harbor Laboratory(1982). Two widely utilized yeast for production of eukaryotic proteinsare Saccharomyces cerevisiae and Pichia pastoris. Vectors, strains, andprotocols for expression in Saccharomyces and Pichia are known in theart and available from commercial suppliers (e.g., Invitrogen). Suitablevectors usually have expression control sequences, such as promoters,including 3-phosphoglycerate kinase or alcohol oxidase, and an origin ofreplication, termination sequences and the like as desired.

A protein of the present invention, once expressed, can be isolated fromyeast by lysing the cells and applying standard protein isolationtechniques to the lysates. The monitoring of the purification processcan be accomplished by using Western blot techniques or radioimmunoassayof other standard immunoassay techniques.

The proteins of the present invention can also be constructed usingnon-cellular synthetic methods. Solid phase synthesis of proteins ofless than about 50 amino acids in length may be accomplished byattaching the C-terminal amino acid of the sequence to an insolublesupport followed by sequential addition of the remaining amino acids inthe sequence. Techniques for solid phase synthesis are described byBarany and Merrifield, Solid-Phase Peptide Synthesis, pp. 3-284 in ThePeptides: Analysis, Synthesis, Biology. Vol. 2: Special Methods inPeptide Synthesis, Part A.; Merrifield et al., J. Am. Chem. Soc.85:2149-2156 (1963), and Stewart et al., Solid Phase Peptide Synthesis,2nd ed., Pierce Chem. Co., Rockford, Ill. (1984). Proteins of greaterlength may be synthesized by condensation of the amino and carboxytermini of shorter fragments. Methods of forming peptide bonds byactivation of a carboxy terminal end (e.g., by the use of the couplingreagent N,N′-dicycylohexylcarbodiimide) is known to those of skill.

The proteins of this invention may be purified to substantial purity bystandard techniques well known in the art, including detergentsolubilization, selective precipitation with such substances as ammoniumsulfate, column chromatography, immunopurification methods, and others.See, for instance, R. Scopes, Protein Purification: Principles andPractice, Springer-Verlag: New York (1982); Deutscher, Guide to ProteinPurification, Academic Press (1990). For example, antibodies may beraised to the proteins as described herein. Purification from E. colican be achieved following procedures described in U.S. Pat. No.4,511,503. Detection of the expressed protein is achieved by methodsknown in the art and include, for example, radioimmunoassays, Westernblotting techniques or immunoprecipitation.

The present invention further provides a method for modulating (i.e.,increasing or decreasing) the concentration or composition of thepolypeptides of the present invention in a plant or part thereof.Modulation of the polypeptides can be effected by increasing ordecreasing the concentration and/or the composition of the polypeptidesin a plant. The method comprises transforming a plant cell with anexpression cassette comprising a polynucleotide of the present inventionto obtain a transformed plant cell, growing the transformed plant cellunder plant forming conditions, and inducing expression of thepolynucleotide in the plant for a time sufficient to modulateconcentration and/or composition of the polypeptides in the plant orplant part.

In some embodiments, the content and/or composition of polypeptides ofthe present invention in a plant may be modulated by altering, in vivoor in vitro, the promoter of a non-isolated gene of the presentinvention to up- or down-regulate gene expression. In some embodiments,the coding regions of native genes of the present invention can bealtered via substitution, addition, insertion, or deletion to decreaseactivity of the encoded enzyme. See, e.g., Kmiec, U.S. Pat. No.5,565,350; Zarling et al., PCT/US93/03868.

In particular, modulating cell cycle proteins are expected to provide apositive growth advantage and increase crop yield. Cell cycle nucleicacids can be adducted to a second nucleic acid sequence encoding aDNA-binding domain, for use in two-hybrid systems to identifyinteracting proteins. It is expected that modulating the level of cellcycle protein, i.e. overexpression in conjunction with overexpression ofG1/S transition-stimulating genes, will increase endoreduplication.Endoreduplication is expected to increase the size of the seed, the sizeof the endosperm and the amount of protein in the seed.

An isolated nucleic acid (e.g., a vector) comprising a promoter sequencecan be transfected into a plant cell. Subsequently, a plant cellcomprising the isolated nucleic acid is selected for by means known tothose of skill in the art such as, but not limited to, Southern blot,DNA sequencing, or PCR analysis using primers specific to the promoterand to the nucleic acid and detecting amplicons produced therefrom. Aplant or plant part altered or modified by the foregoing embodiments isgrown under plant forming conditions for a time sufficient to modulatethe concentration and/or composition of polypeptides of the presentinvention in the plant. Plant forming conditions are well known in theart.

In general, concentration of the polypeptides is increased or decreasedby at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% relativeto a native control plant, plant part, or cell lacking theaforementioned expression cassette. Modulation in the present inventionmay occur during and/or subsequent to growth of the plant to the desiredstage of development.

Modulating nucleic acid expression temporally and/or in particulartissues can be controlled by employing the appropriate promoter operablylinked to a polynucleotide of the present invention in, for example,sense or antisense orientation as discussed in greater detail above.Induction of expression of a polynucleotide of the present invention canalso be controlled by exogenous administration of an effective amount ofinducing compound. Inducible promoters and inducing compounds thatactivate expression from these promoters are well known in the art.

In preferred embodiments, the polypeptides of the present invention aremodulated in monocots or dicots, preferably corn, soybean, sunflower,safflower, sorghum, canola, wheat, alfalfa, cotton, rice, barley andmillet.

Means of detecting the proteins of the present invention are notcritical aspects of the present invention. In a preferred embodiment,the proteins are detected and/or quantified using any of a number ofwell-recognized immunological binding assays (see, e.g., U.S. Pat. Nos.4,366,241; 4,376,110; 4,517,288; and 4,837,168). For a review of thegeneral immunoassays, see also Methods in Cell Biology, Vol. 37:Antibodies in Cell Biology, Asai, Ed., Academic Press, Inc. New York(1993); Basic and Clinical Immunology 7th Edition, Stites & Terr, Eds.(1991). Moreover, the immunoassays of the present invention can beperformed in any of several configurations, e.g., those reviewed inEnzyme Immunoassay, Maggio, Ed., CRC Press, Boca Raton, Fla. (1980);Tijan, Practice and Theory of Enzyme Immunoassays, Laboratory Techniquesin Biochemistry and Molecular Biology, Elsevier Science Publishers B.V., Amsterdam (1985); Harlow and Lane, supra; Immunoassay: A PracticalGuide, Chan, Ed., Academic Press, Orlando, Fla. (1987); Principles andPractice of Immunoassays, Price and Newman Eds., Stockton Press, NY(1991); and Non-isotopic Immunoassays, Ngo, Ed., Plenum Press, NY(1988).

Typical methods for detecting proteins include Western blot (immunoblot)analysis, analytic biochemical methods such as electrophoresis,capillary electrophoresis, high performance liquid chromatography(HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography,and the like, and various immunological methods such as fluid or gelprecipitin reactions, immunodiffusion (single or double),immunoelectrophoresis, radioimmunoassays (RIAs), enzyme-linkedimmunosorbent assays (ELISAs), immunofluorescent assays, and the like.

Non-radioactive labels are often attached by indirect means. Generally,a ligand molecule (e.g., biotin) is covalently bound to the molecule.The ligand then binds to an anti-ligand (e.g., streptavidin) moleculethat is either inherently detectable or covalently bound to a signalsystem, such as a detectable enzyme, a fluorescent compound, or achemiluminescent compound. A number of ligands and anti-ligands can beused. Where a ligand has a natural anti-ligand, for example, biotin,thyroxine, and cortisol, it can be used in conjunction with the labeled,naturally occurring anti-ligands. Alternatively, any haptenic orantigenic compound can be used in combination with an antibody.

The molecules can also be conjugated directly to signal generatingcompounds, e.g., by conjugation with an enzyme or fluorophore. Enzymesof interest as labels will primarily be hydrolases, particularlyphosphatases, esterases and glycosidases, or oxidoreductases,particularly peroxidases. Fluorescent compounds include fluorescein andits derivatives, rhodamine and its derivatives, dansyl, umbelliferone,etc. Chemiluminescent compounds include luciferin, and2,3-dihydrophthalazinediones, e.g., luminol. For a review of variouslabeling or signal producing systems which may be used, see, U.S. Pat.No. 4,391,904, which is incorporated herein by reference.

Some assay formats do not require the use of labeled components. Forinstance, agglutination assays can be used to detect the presence of thetarget antibodies. In this case, antigen-coated particles areagglutinated by samples comprising the target antibodies. In thisformat, none of the components need be labeled and the presence of thetarget antibody is detected by simple visual inspection.

The proteins of the present invention can be used for identifyingcompounds that bind to (e.g., substrates), and/or increase or decrease(i.e., modulate) the enzymatic activity of, catalytically activepolypeptides of the present invention. The method comprises contacting apolypeptide of the present invention with a compound whose ability tobind to or modulate enzyme activity is to be determined. The polypeptideemployed will have at least 20%, preferably at least 30% or 40%, morepreferably at least 50% or 60%, and most preferably at least 70% or 80%of the specific activity of the native, full-length polypeptide of thepresent invention (e.g., enzyme). Methods of measuring enzyme kineticsare well known in the art. See, e.g., Segel, Biochemical Calculations,2nd ed., John Wiley and Sons, New York (1976).

Antibodies can be raised to a protein of the present invention,including individual, allelic, strain, or species variants, andfragments thereof, both in their naturally occurring (full-length) formsand in recombinant forms. Additionally, antibodies are raised to theseproteins in either their native configurations or in non-nativeconfigurations. Anti-idiotypic antibodies can also be generated. Manymethods of making antibodies are known to persons of skill.

In some instances, it is desirable to prepare monoclonal antibodies fromvarious mammalian hosts, such as mice, rodents, primates, humans, etc.Description of techniques for preparing such monoclonal antibodies arefound in, e.g., Basic and Clinical Immunology, 4th ed., Stites et al.,Eds., Lange Medical Publications, Los Altos, Calif., and referencescited therein; Harlow and Lane, Supra; Goding, Monoclonal Antibodies:Principles and Practice, 2nd ed., Academic Press, New York, N.Y. (1986);and Kohler and Milstein, Nature 256:495-497 (1975).

Other suitable techniques involve selection of libraries of recombinantantibodies in phage or similar vectors (see, e.g., Huse et al., Science246:1275-1281 (1989); and Ward, et al., Nature 341:544-546 (1989); andVaughan et al., Nature Biotechnology, 14:309-314 (1996)). Alternatively,high avidity human monoclonal antibodies can be obtained from transgenicmice comprising fragments of the unrearranged human heavy and lightchain Ig loci (i.e., minilocus transgenic mice). Fishwild et al., NatureBiotech., 14:845-851 (1996). Also, recombinant immunoglobulins may beproduced. See, Cabilly, U.S. Pat. No. 4,816,567; and Queen et al., Proc.Nat'l Acad. Sci. 86:10029-10033 (1989).

The antibodies of this invention can be used for affinity chromatographyin isolating proteins of the present invention, for screening expressionlibraries for particular expression products such as normal or abnormalprotein or for raising anti-idiotypic antibodies which are useful fordetecting or diagnosing various pathological conditions related to thepresence of the respective antigens.

Frequently, the proteins and antibodies of the present invention will belabeled by joining, either covalently or non-covalently, a substance,which provides for a detectable signal. A wide variety of labels andconjugation techniques are known and are reported extensively in boththe scientific and patent literature. Suitable labels includeradionucleotides, enzymes, substrates, cofactors, inhibitors,fluorescent moieties, chemiluminescent moieties, magnetic particles, andthe like.

Transfection/Transformation of Cells

The method of transformation/transfection is not critical to the presentinvention; various methods of transformation or transfection arecurrently available. As newer methods are available to transform cropsor other host cells they may be directly applied. Accordingly, a widevariety of methods have been developed to insert a DNA sequence into thegenome of a host cell to obtain the transcription and/or translation ofthe sequence to effect phenotypic changes in the organism. Thus, anymethod that provides for efficient transformation/transfection may beemployed.

A DNA sequence coding for the desired polynucleotide of the presentinvention can be used to construct an expression cassette that can beintroduced into the desired plant. Isolated nucleic acid acids of thepresent invention can be introduced into plants according techniquesknown in the art. Generally, expression cassettes as described above andsuitable for transformation of plant cells are prepared.

Techniques for transforming a wide variety of higher plant species arewell known and described in the technical, scientific, and patentliterature. See, for example, Weising et al., Ann. Rev. Genet.22:421-477 (1988). For example, the DNA construct may be introduceddirectly into the genomic DNA of the plant cell using techniques such aselectroporation, PEG poration, particle bombardment, silicon fiberdelivery, or microinjection of plant cell protoplasts or embryogeniccallus. See, e.g., Tomes, et al., Direct DNA Transfer into Intact PlantCells Via Microprojectile Bombardment. pp. 197-213 in Plant Cell, Tissueand Organ Culture, Fundamental Methods. eds. O. L. Gamborg and G. C.Phillips. Springer-Verlag Berlin Heidelberg N.Y., 1995. The introductionof DNA constructs using polyethylene glycol precipitation is describedin Paszkowski et al., Embo J. 3:2717-2722 (1984). Electroporationtechniques are described in Fromm et al., Proc. Natl. Acad. Sci. 82:5824(1985). Ballistic transformation techniques are described in Klein etal., Nature 327:70-73 (1987).

Alternatively, the DNA constructs may be combined with suitable T-DNAflanking regions and introduced into a conventional Agrobacteriumtumefaciens host vector. The virulence functions of the Agrobacteriumtumefaciens host will direct the insertion of the construct and adjacentmarker into the plant cell DNA when the cell is infected by thebacteria. See, U.S. Pat. No. 5,981,840. Agrobacteriumtumefaciens-meditated transformation techniques are well described inthe scientific literature. See, for example Horsch et al., Science233:496-498 (1984), and Fraley et al., Proc. Natl. Acad. Sci. 80:4803(1983). For instance, Agrobacterium transformation of maize is describedin WO 98/32326. Agrobacterium transformation of soybean is described inU.S. Pat. No. 5,563,055.

Other methods of transfection or transformation include (1)Agrobacterium rhizogenes-mediated transformation (see, e.g.,Lichtenstein and Fuller In: Genetic Engineering, Vol. 6, P W J Rigby,Ed., London, Academic Press, 1987; and Lichtenstein, C. P., and Draper,J,. In: DNA Cloning, Vol. II, D. M. Glover, Ed., Oxford, IRI Press,1985), Application PCT/US87/02512 (WO 88/02405 published Apr. 7, 1988)describes the use of A. rhizogenes strain A4 and its Ri plasmid alongwith A. tumefaciens vectors pARC8 or pARC16 (2) liposome-mediated DNAuptake (see, e.g., Freeman et al., Plant Cell Physiol. 25:1353, (1984)),(3) the vortexing method (see, e.g., Kindle, Proc. Natl. Acad. Sci. USA87:1228, (1990)).

DNA can also be introduced into plants by direct DNA transfer intopollen as described by Zhou et al., Methods in Enzymology, 101:433(1983); D. Hess, Intern Rev. Cytol., 107:367 (1987); Luo et al., PlaneMol. Biol. Reporter, 6:165 (1988). Expression of polypeptide codingpolynucleotides can be obtained by injection of the DNA intoreproductive organs of a plant as described by Pena et al., Nature,325:274 (1987). DNA can also be injected directly into the cells ofimmature embryos and the rehydration of desiccated embryos as describedby Neuhaus et al., Theor. Appl. Genet., 75:30 (1987); and Benbrook etal., in Proceedings Bio Expo 1986, Butterworth, Stoneham, Mass., pp.27-54 (1986).

Animal and lower eukaryotic (e.g., yeast) host cells are competent orrendered competent for transfection by various means. There are severalwell-known methods of introducing DNA into animal cells. These include:calcium phosphate precipitation, fusion of the recipient cells withbacterial protoplasts containing the DNA, treatment of the recipientcells with liposomes containing the DNA, DEAE dextran, electroporation,biolistics, and micro-injection of the DNA directly into the cells. Thetransfected cells are cultured by means well known in the art. Kuchler,R. J., Biochemical Methods in Cell Culture and Virology, Dowden,Hutchinson and Ross, Inc. (1977).

Stable transformation of some gene products into recipient cells isproblematic for regulatory and other reasons. Therefore, it is desirableto transiently express proteins in transformed cells. UsingArgrobacterium as a protein vector for transient expression ispotentially simpler and would deliver a selected protein and a desiredtransgene to the same cell simultaneously.

Certain species of symbiotic micro-organisms are known to transfer T-DNAinto recipient cells by a mechanism similar to bacterial conjugation.T-DNA traverses the bacterial membranes, the cell wall and cellmembranes, and the nuclear membrane before integrating into the hostgenome through illegitimate recombination. Numerous bacterial proteinsare also included in these processes and have been characterized. Amongthese proteins are at least three gene products from Agrobacterium:VirD2, VirE2, and VirF which are transcribed from the virulence regionof the Ti plasmid and transferred directly into plant cells.

VirD2 encodes a multifunctional protein which participates in theendonucleolytic cleavage of the T-DNA border sequences, the ligation ofthe left border nick for replacement strand synthesis, nuclear import ofthe T-complex, and precise integration of the 5′ end of T-DNA into thehost genome. VirD2 establishes a covalent association with the T-DNAbetween a specific right-border (RB) nucleotide and Tyr-29 of theprotein.

VirE2 encodes a multifunctional protein that has single-stranded DNAbinding (SSB) activity and coats the T-strand. VirE2 is also likely tobe involved both in nuclear import and with the integration offull-length T-DNA into the host genome. VirE2 is the most abundant ofVir proteins with 350 to 700 copies thought to be required to coat a 20kb T-strand.

The function of the VirF gene product is unknown. The coding sequence ispresent in octopine strains but not in nopaline strains. Complementationof nopaline strains or VirF mutants of octopine strains extends hostrange.

VirE2 is the most preferred product for use as a delivery protein fusionvector. First, it is produced in high abundance. Second, it can betransmitted separately from the T-strand to plant cells. VirD2, incontrast, is covalently associated with the T-strand. Third, VirE2 hasbeen studied intensively and functional domains are known. Relativelylittle information is available for VirF.

Proteins delivered from Agrobacterium plasmids into plant cells are inthe form of fusions with the Agrobacterium virulence proteins. Fusionsare constructed between a selected gene and genes for bacterialvirulence proteins such as VirE2, VirD2, or VirF which are locatedoutside the T-DNA borders. This leaves an expression cassette within theborders available for genes that are to be stably transformed. Fusionsare constructed to retain both those properties of bacterial virulenceproteins required to mediate delivery into plant cells and the selectedactivity required for altering cell function. This method ensures a highfrequency of simultaneous co-delivery of T-DNA and the functionalselected protein into the same host cell.

An example is the delivery of a VirE2::“cell cycle protein” fusion toplant cells. Several candidate genes that might stimulate the G1→Stransition are available. Examples are well known in the art such ascyclins (P. W. Doerner, Cell Cycle Regulation in Plants, Plant Physiol.(1994) 106:823-827.), and the gemini virus RepA gene (U.S. Ser. No.09/257,131). The promotion of S phase by transient “expression” ofselected cell cycle proteins may enhance integration of the coresidentT-DNA. Other fusion partners and applications of protein delivery areconceivable.

The method can be used to test the efficacy of visible selectablemarkers such as GFP (Haseloff et al, Trends in Genetics 11(8):328-329(1995), GUS (beta-gluconronidase), and Luciferase, (Visser et al.,Biochemistry 24(6):1489-1496 (1985). Or the visible markers could beused in the system to test changes in protocols that would enhancetransfer of molecules to various plant cells, or cells or tissues ofrecalcitrant species.

Using the method with selected proteins such as Bcl-2 (Pegoraro et al.,Proc. Nat. Ac. Sci 81(22):7166-7170 (1984), or IAP (inhibitor ofapoptosis) (Crook, et al., Journ. Vir. 67(4):2168-2174 (1993), wouldreduce the tendency of recently transformed cells to undergo programmedcell death, and in the process increase transgene integration andoverall transformation frequencies.

Fusing the delivery protein to genes such as fus3 (Elion et al., Cell60(4):649-664 (1990), CLAVATA (Clark et al., Development (Cambridge)122(5):1567-1575 (1996), KNOTTED-1 (Lowe et al., Genetics 132(3):813-822(1992), or pk1 (Ogas et al, Science (Washington D.C.) 277(5322):91-94(1997) would commit cells and cell lineages to a desired developmentalfate such as meristem development or stimulating embryo development.

Introduction of a site-specific recombinase protein system such asFLP/RFT (U.S. Ser. No. 08,972,258) or Cre/loxP (Abremski-K. et al.,Jour. Mol. Bio. 184(2):211-220, 1985) into plant cells could be used tocatalyze a variety of recombination-mediated alterations. For example,sequence excision could be used to remove one transgene while activatinga second. Recombinase-mediated integration, gene replacement and genomicexchanges could also be mediated through introduction of such functionalfusion proteins.

The method can also be practiced with other strains of bacteria known todeliver protein into cells. Examples are: Rhizobium sp., Phyllobacteriumsp., or any other bacterium of the Rhizobiaceae taxa that transferproteins to recipient cells.

The method could be extended to employ multiple delivery protein fusionson the same, or coresident, binaries. This would conceivably allow thetransient activity of “protein cocktails” mediating complex functions orpathways related to transformation objectives.

The Argrobacterium strategy is potentially simpler than methods toachieve transient-only expression using current direct delivery methodssuch as microinjection, bombardment, electroporation or silica fibermethods.

Transgenic Plant Regeneration

Transformed plant cells which are derived by any of the abovetransformation techniques can be cultured to regenerate a whole plantwhich possesses the transformed genotype. Such regeneration techniquesoften rely on manipulation of certain phytohormones in a tissue culturegrowth medium, typically relying on a biocide and/or herbicide markerthat has been introduced together with a polynucleotide of the presentinvention. For transformation and regeneration of maize see, Gordon-Kammet al., The Plant Cell, 2:603-618 (1990).

Plants cells transformed with a plant expression vector can beregenerated, e.g., from single cells, callus tissue or leaf discsaccording to standard plant tissue culture techniques. It is well knownin the art that various cells, tissues, and organs from almost any plantcan be successfully cultured to regenerate an entire plant. Plantregeneration from cultured protoplasts is described in Evans et al.,Protoplasts Isolation and Culture, Handbook of Plant Cell Culture,Macmillan Publishing Company, New York, pp. 124-176 (1983); and Binding,Regeneration of Plants, Plant Protoplasts, CRC Press, Boca Raton, pp.21-73 (1985).

The regeneration of plants containing the foreign gene introduced byArgrobacterium can be achieved as described by Horsch et al., Science,227:1229-1231 (1985) and Fraley et al., Proc. Natl. Acad. Sci. U.S.A.,80:4803 (1983). This procedure typically produces shoots within two tofour weeks and these transformant shoots are then transferred to anappropriate root-inducing medium containing the selective agent and anantibiotic to prevent bacterial growth. Transgenic plants of the presentinvention may be fertile or sterile.

Regeneration can also be obtained from plant callus, explants, organs,or parts thereof. Such regeneration techniques are described generallyin Klee et al., Ann. Rev. of Plant Phys. 38: 467-486 (1987). Theregeneration of plants from either single plant protoplasts or variousexplants is well known in the art. See, for example, Methods for PlantMolecular Biology, A. Weissbach and H. Weissbach, eds., Academic Press,Inc., San Diego, Calif. (1988). For maize cell culture and regenerationsee generally, The Maize Handbook, Freeling and Walbot, Eds., Springer,N.Y. (1994); Corn and Corn Improvement, 3^(rd) edition, Sprague andDudley Eds., American Society of Agronomy, Madison, Wis. (1988).

One of skill will recognize that after the expression cassette is stablyincorporated in transgenic plants and confirmed to be operable, it canbe introduced into other plants by sexual crossing. Any of a number ofstandard breeding techniques can be used, depending upon the species tobe crossed.

In vegetatively propagated crops, mature transgenic plants can bepropagated by the taking of cuttings or by tissue culture techniques toproduce multiple identical plants. Selection of desirable transgenics ismade and new varieties are obtained and propagated vegetatively forcommercial use. In seed propagated crops, mature transgenic plants canbe self-crossed to produce a homozygous inbred plant. The inbred plantproduces seed containing the newly introduced heterologous nucleic acid.These seeds can be grown to produce plants that would produce theselected phenotype.

Parts obtained from the regenerated plant, such as flowers, seeds,leaves, branches, fruit, and the like are included in the invention,provided that these parts comprise cells comprising the isolated nucleicacid of the present invention. Progeny and variants, and mutants of theregenerated plants are also included within the scope of the invention,provided that these parts comprise the introduced nucleic acidsequences.

Transgenic plants expressing a selectable marker can be screened fortransmission of the nucleic acid of the present invention by, forexample, standard immunoblot and DNA detection techniques. Transgeniclines are also typically evaluated on levels of expression of theheterologous nucleic acid. Expression at the RNA level can be determinedinitially to identify and quantitate expression-positive plants.Standard techniques for RNA analysis can be employed and include PCRamplification assays using oligonucleotide primers designed to amplifyonly the heterologous RNA templates and solution hybridization assaysusing heterologous nucleic acid-specific probes. The RNA-positive plantscan then analyzed for protein expression by Western immunoblot analysisusing the specifically reactive antibodies of the present invention. Inaddition, in situ hybridization and immunocytochemistry according tostandard protocols can be done using heterologous nucleic acid specificpolynucleotide probes and antibodies, respectively, to localize sites ofexpression within transgenic tissue. Generally, a number of transgeniclines are usually screened for the incorporated nucleic acid to identifyand select plants with the most appropriate expression profiles.

A preferred embodiment is a transgenic plant that is homozygous for theadded heterologous nucleic acid; i.e., a transgenic plant that containstwo added nucleic acid sequences, one gene at the same locus on eachchromosome of a chromosome pair. A homozygous transgenic plant can beobtained by sexually mating (selfing) a heterozygous transgenic plantthat contains a single added heterologous nucleic acid, germinating someof the seed produced and analyzing the resulting plants produced foraltered expression of a polynucleotide of the present invention relativeto a control plant (i.e., native, non-transgenic). Backcrossing to aparental plant and out-crossing with a non-transgenic plant are alsocontemplated.

The present invention provides a method of genotyping a plant comprisinga polynucleotide of the present invention. Genotyping provides a meansof distinguishing homologs of a chromosome pair and can be used todifferentiate segregants in a plant population. Molecular marker methodscan be used for phylogenetic studies, characterizing geneticrelationships among crop varieties, identifying crosses or somatichybrids, localizing chromosomal segments affecting monogenic traits, mapbased cloning, and the study of quantitative inheritance. See, e.g.,Plant Molecular Biology: A Laboratory Manual, Chapter 7, Clark, Ed.,Springer-Verlag, Berlin (1997). For molecular marker methods, seegenerally, The DNA Revolution by Andrew H. Paterson 1996 (Chapter 2) in:Genome Mapping in Plants (ed. Andrew H. Paterson) by Academic Press/R.G. Landis Company, Austin, Tex., pp.7-21.

The particular method of genotyping in the present invention may employany number of molecular marker analytic techniques such as, but notlimited to, restriction fragment length polymorphisms (RFLPs). RFLPs arethe product of allelic differences between DNA restriction fragmentscaused by nucleotide sequence variability. Thus, the present inventionfurther provides a means to follow segregation of a gene or nucleic acidof the present invention as well as chromosomal sequences geneticallylinked to these genes or nucleic acids using such techniques as RFLPanalysis.

Plants that can be used in the method of the invention includemonocotyledonous and dicotyledonous plants. Preferred plants includecorn, soybean, sunflower, safflower, sorghum, canola, wheat, alfalfa,cotton, rice, barley and millet. Seeds derived from plants regeneratedfrom transformed plant cells, plant parts or plant tissues, or progenyderived from the regenerated transformed plants, may be used directly asfeed or food, or further processing may occur.

The present nucleic acids and proteins have many uses. They can be usedto identify other interacting proteins involved in cell cycleregulation. They can be used to provide antigenic proteins. Altering theexpression of the present nucleic acids and proteins provides a methodfor modulating cell division, especially for increasing the number ofcells undergoing cell division. This has been found useful in improvingtransformation efficiency.

Use in Two-Hybrid Systems

An important utility for the maize CycE genes that have been cloned inthe genetic approach of using a two-hybrid system to identifyinteracting proteins (i.e. proteins that specifically interact with theCycE gene-encoded products. This method, typically done using the yeastSaccharomyces cerevisiae, exploits the fact that a functionaltranscription factor can be separated into two components; a DNA-bindingfactor and an activation domain, which when held together non-covalentlywill still bind DNA and activate transcription.

The test system is constructed as follows: a DNA-binding domain islocalized 5′ to a reporter gene, for example luciferase, and thiscassette is transformed into a yeast strain. The nucleic acid sequencefor the DNA-binding domain of the transcriptional factor is ligated tothe gene (or partial gene sequence) being used as bait. Expression ofthis DNA-binding domain-bait fusion is driven, for example by the yeastadh1 promoter. A “library” of gene-fusions is also produced, using theactivation domain of the transcriptional factor fused to genes (or genefragments) from an expression library of interest (referred to as theactivation domain hybrid). Expression of the activation domain hybridsis also accomplished, for example, using the yeast adh1 promoter.

To perform the two-hybrid screen, plasmids encoding the DNA-bindingdomain hybrid and a library of activation domain hybrids are introduced(sequentially or simultaneously) into a yeast strain already containingthe inactive reporter. Transformed yeast in which the activation domainhybrid specifically binds to the DNA-binding domain hybrid will expressluciferase. Positives are further characterized by sequence analysis,and further tests of relevance of biological interactions.

Commonly used DNA-binding domains include those from lexa protein in E.coli, and the Ga14 protein in yeast. Likewise, commonly used activationdomains include B42 (bacterial) and Ga14 (yeast). For details, seeHannon G, and Bartel P, Identification of interacting proteins using thetwo-hybrid system, Methods Mol. Cellular Biol. 5:289-297 (1995).

The nucleic acids and proteins of the present invention modulate therate of cell division and the total number of cells. Increasing thetotal number of cells in a plant is expected to increase crop yield. Itis also expected that the present invention provides a method formodulating plant height or size. The present invention provides a methodfor modulating cell growth. In particular it is expected that thepresent inventive nucleic acids and proteins will provide a method forincreasing the growth rate and providing a positive growth advantage ina plant. The present invention is expected to provide a method forenhancing or inhibiting organ growth, for example seed, root, shoot,ear, tassel, stalk, pollen, stamen. Therefore, the nucleic acids andproteins of the present invention may provide a method for producingorgan ablation, such as for parthenocarpic fruits or male sterileplants. The nucleic acids and proteins can be used to increase thenumber of pods per plant and/or seeds/pod or ear. The nucleic acids andproteins of the present invention may provide a method for altering thelag time in seed development. The nucleic acids of the present inventionare expected to provide a method for improving in cells the response toenvironmental stress such as drought, heat, or cold.

The nucleic acids and proteins of the present invention provide a methodfor enhancing embryogenic response, i.e. size or growth rate. They arealso expected to provide a method for increasing callus induction. Thenucleic acids and proteins of the present invention should provide amethod for positive selection and/or increasing plant regeneration. Thenucleic acids and proteins of the present invention may provide a methodfor altering the percent of cells that are arrested or for altering theamount of time a cell spends in a particular cell cycle, i.e. in G1 orG0 stages of the cell cycle. The nucleic acids and proteins of thepresent invention should provide hormone independent cell growth. Thenucleic acids and proteins of the present invention may also provide amethod for increasing growth rate of cells in bioreactors.

All cited publications are incorporated herein by reference.

Although the present invention has been described in some detail by wayof illustration and example for purposes of clarity of understanding, itwill be obvious that certain changes and modifications may be practicedwithin the scope of the appended claims.

EXAMPLES Example 1 Isolation of Maize CycE Genes

Total RNA was isolated from corn tissues with TRIzol Reagent (LifeTechnology Inc. Gaithersburg, Md.) using a modification of the guanidineisothiocyanate/acid-phenol procedure described by Chomczynski and Sacchi[Chomczynski, P., and Sacchi, N., Anal. Biochem. 162, 156 (1987)]. Inbrief, plant tissue samples were pulverized in liquid nitrogen beforethe addition of the TRIzol Reagent, and then were further homogenizedwith a mortar and pestle. Addition of chloroform followed bycentrifugation was conducted for separation of an aqueous phase and anorganic phase. The total RNA was recovered by precipitation withisopropyl alcohol from the aqueous phase.

Poly(A)+ RNA Isolation

The selection of poly(A)+ RNA from total RNA was performed usingPolyATract system (Promega Corporation. Madison, Wis.). In brief,biotinylated oligo(dT) primers were used to hybridize to the 3′ poly(A′)tails on mRNA. The hybrids were captured using streptavidin coupled toparamagnetic particles and a magnetic separation stand. The mRNA waswashed using high stringency conditions and eluted using RNAase-freedeionized water.

cDNA Library Construction:

cDNA synthesis was performed and unidirectional cDNA libraries wereconstructed using the SuperScript Plasmid System (Life Technology Inc.Gaithersburg, Md.). The first stand of cDNA was synthesized by primingan oligo(dT) primer containing a Not I site. The reaction was catalyzedby SuperScript Reverse Transcriptase II at 45° C. The second strand ofcDNA was labeled with alpha-³²P-dCTP and a portion of the reaction wasanalyzed by agarose gel electrophoresis to determine cDNA sizes. cDNAmolecules smaller than 500 base pairs and unligated adapters wereremoved by Sephacryl-S400 chromatography. The selected cDNA moleculeswere ligated into pSPORT1 vector in between Not I and Sal I sites.Mitotically active tissues from Zea mays were employed, including suchsources as shoot cultures, immature inflorescences (tassel and ear) aswell as other sources of vegetative meristems.

Sequencing Template Preparation:

Individual colonies were picked and DNA was prepared either by PCR withM13 forward primers and M13 reverse primers, or by plasmid isolation.All the cDNA clones were initially sequenced using M13 reverse primers.As additional fragments of the genes were discovered, new sequencingprimers were designed.

PROTOCOLS, Murray (ed.), pages 271-281 (Humana Press, Inc. 1991).Functional fragments of the cell cycle protein are identified by theirability, upon introduction to cells, to stimulate the G1 to S-phasetransition, which is manifested by increased DNA replication in apopulation of cells and by increased cell division rates.

5′-RACE

Library RACE was performed using several of Pioneer's maize libraries.5′ RACE was done using a cDNA library constructed from leaves and stemsof maize plants at the three-leaf stage. The principal of 5′ RACE isdescribed in detail in numerous publications such as: Frohman M. A.,1993, Rapid Amplification of Complementary DNA Ends for Generation ofFull-Length Complementary DNAs: Thermal RACE. In: Methods in Enzymology,vol. 28, pp 340-356. Detailed procedure can be found in the ClonTechMarathon cloning manual.

Example 2 Using CycE's in a Two-hybrid System to Identify Maize CellCycle Genes

CycE gene expression during the G1→S transition and early S-phase playsa prominent role in progression through the cell cycle. The proteinsencoded by the CycE gene family are an important part of the complexthat binds and phosphorylates retinoblastoma-associated gene familymembers. In turn, Rb releases E2F and this transcription factor startsthe cascade of events leading to DNA replication. As such, the CycEgenes and their encoded proteins can be used to identify other cellcycle regulatory proteins. This can be done using the CycE gene as bait(the target fused to the DNA-binding domain) in a yeast two-hybridscreen. Methods for two-hybrid library construction, cloning of thereporter gene, cloning of the DNA-binding and activation domain hybridgene cassettes, yeast culture, and transformation of the yeast are alldone according to well-established methods (see Sambrook et al., 1990;Ausubel et al., 1990; Hannon and Bartels, 1995). Using this method, Zeamays Cdk2 and Rb genes are identified as components of the activationdomain hybrid, and are confirmed through further sequence analysis.Similarly, inhibitors of the Cdk2/CycE complex such as the CIP/KIPfamily (p21, p27, p57), and enhancers of the Cdk2/CycE complex similarto p37 are identified.

Example 3 CycE-bound Affinity Columns for Identifying Cdk2 Proteins andtheir Encoding Genes

Purified recombinant CycE protein can be immobilized on a matrix via acovalent crosslinking or affinity purification as described supra. Thismatrix can then be used to pull-down proteins that interact with CycEproteins, inter alia, cyclin-dependent kinase. CDK activity can then beassessed by measuring the addition of radioactive phosphorus toprotein-substrates and CDK protein levels determined by immunoassay.Additionally, this can be used to purify the CDK activity present indifferent plant tissues and protein fractions. The presence and level ofother CycE interacting proteins can also be determined on the basis ofimmunological assay, activity quantification, SDS-PAGE analysis andother methods. These measures can then be correlated with thereproductive state, capacity for division, developmental stage, or thequality of different samples. A CycE nucleic acid can also be adductedto a second nucleic acid sequence encoding a DNA-binding domain in orderto identify CycE interacting proteins.

Example 4 Using the CycE Gene to Improve Maize Transformation

Delivery of the ZmCycE gene can be accomplished through numerouswell-established methods for plant cells, including for example particlebombardment, sonication, PEG treatment or electroporation ofprotoplasts, electroporation of intact tissue, silica-fiber methods,microinjection or Agrobacterium-mediated transformation. Using one ofthe above methods, DNA is introduced into maize cells capable of growthon suitable maize culture medium. Such competent cells can be from maizesuspension culture, callus culture on solid medium, freshly isolatedimmature embryos or meristem cells. Immature embryos of the Hi-IIgenotype are used as the target for co-delivery of these two plasmids.For target tissues receiving the CycE expression cassette,transformation frequency is improved.

Particle-mediated DNA Delivery

The CycE gene (ZmCycE) is cloned into a cassette with a constitutivepromoter (the maize ubiquitin promoter, UBI, including the firstubiquitin intron) and a 3′ sequence from the potato proteinase inhibitor(pinII). Particle bombardment is used to introduce theUBI::ZmCycE::pinII-containing plasmid along with aUBI::PAT˜GFP::pinII-containing plasmid (which, when expressed produces afunctional PAT˜GFP fusion protein which confers bialaphos resistance andgreen fluorescence) into maize cells capable of growth on suitable maizeculture medium. Such competent cells can be from maize suspensionculture, callus culture on solid medium, freshly isolated immatureembryos or meristem cells. Immature embryos of the Hi-II genotype areused as the target for co-delivery of these two plasmids. Ears areharvested at approximately 10 days post-pollination, and 1.2-1.5 mmimmature embryos are isolated from the kernels, and placedscutellum-side down on maize culture medium.

The immature embryos are bombarded from 18-72 hours after beingharvested from the ear. Between 6 and 18 hours prior to bombardment, theimmature embryos are placed on medium with additional osmoticum (MSbasal medium, Musashige and Skoog, 1962, Physiol. Plant 15:473-497, with0.25 M sorbitol). The embryos on the high-osmotic medium are used as thebombardment target, and are left on this medium for an additional 18hours after bombardment.

For particle bombardment, plasmid DNA (described above) is precipitatedonto 1.8 μm tungsten particles using standard CaCl₂-spermidine chemistry(see, for example, Klein et al., 1987, Nature 327:70-73). Each plate isbombarded once at 600 PSI, using a DuPont Helium Gun (Lowe et al., 1995,Bio/Technol 13:677-682). For typical media formulations used for maizeimmature embryo isolation, callus initiation, callus proliferation andregeneration of plants, see Armstrong, C., 1994, In “The MaizeHandbook”, M. Freeling and V. Walbot, eds. Springer Verlag, NY, pp663-671.

Selection

Within 1-7 days after particle bombardment, the embryos are moved ontoN6-based culture medium containing 3 mg/l of the selective agentbialaphos. Embryos, and later callus, are transferred to fresh selectionplates every 2 weeks. After the first 14 days post-bombardment, thecalli developing from the immature embryos are screened for GFPexpression using an epifluorescent dissecting-microscope. Typically,(i.e. in the absence of a cell cycle gene) this is too early to observegrowing multicellular transformants. Instead, as typical after such ashort post-bombardment duration, numerous GFP-expressing single-cellsare observed on control embryos (where the UBI::PAT˜GFP::pinII plasmidis introduced alone), but GFP-expressing multicellular clusters are notobserved. It is expected that when UBI::CycE::pinII is included alongwith the UBI::PAT˜GFP::pinII marker, numerous GFP+ multicellularclusters are observed growing from the immature embryos at this sameearly time-point (14 days post-bombardment). The higher number ofrapidly-growing transformants suggests that expression of CycE increasesintegration frequencies (thus higher numbers) and stimulates growth ofthese colonies after integration has occurred (thus, the transformantsare clearly visible at this early juncture).

After 6-8 weeks, transformed calli are recovered. In treatments whereboth the PAT˜GFP gene and CycE are transformed into immature embryos, ahigher number of growing calli are expected on the selective medium andcallus growth is stimulated (relative to treatments with the PAT-GFPgene alone).

Differences in cell cycle profiles are expected in CycE-expressing cellsrelative to control (wild-type) cells. To demonstrate thatover-expression of CycE genes could accelerate cell division, the cellcycle profile of maize calli expressing Ubi::CycE are analyzed using acell sorter (flow cytometry assay). Flow cytometry is a standard methodto study cell cycle, using procedures that are well established in theliterature, as, for example, in Sonea I M et al., Am J Vet Res. 199960(3):346-53.

Briefly, by counting the number of cells that are in G1 phase versus thenumber of cells that are in G2 phase, one can estimate, in a givenpopulation, the percentage of cells that are undergoing cell division.The higher the percentage of cells in G1 phase, the less the number ofcells that are dividing. Under standard culture conditions,approximately 70% of the G1/G2 cells of maize calli are in the G1 phase.In maize calli expressing CycE genes, alterations of the distribution ofcells in the G1 and G2 phases is expected. The frequency of cells in G1declines, and the proportion of the cell population in either S or G2phase increases (indicative of stimulating the progression from G1 intoS phase in CycE-expressing cells). In control calli expressing similarvector genes but lacking a CycE gene, the cell cycle profile remainssimilar to that of the non-treated wild type maize calli.

Calli from the CycE treatment are expected to regenerate easily.Healthy, fertile transgenic plants are grown in the greenhouse. Seed-seton CycE transgenic plants is expected to be similar to control plants,and transgenic progeny are recovered.

It is expected that higher CycE-transgene expression levels improvetransformation. For this bombardment experiment (to be performed in asimilar manner to that described above), Hi-II ears are harvested at 10DAP, and the immature embryos are divided evenly between the 3treatments (125 embryos per treatment). The treatments include ano-cyclin control (UBI::PAT˜GFP::pinII), or the UBI::PAT˜GFP::pinIImarker plus one of two cyclin-expressing plasmids (UBI::CycE ornos::CycE). For this experiment high levels of cyclin expression (UBI)are being compared to low levels (nos) of expression. When the UBIpromoter drives expression, the transformation frequency for the CycEgene is expected to be increased. Placing the CycE gene behind the nospromoter is expected to produce a transformation frequency more similarto the control. It is expected that higher expression levels result incorrespondingly higher recovery of transformants.

It is expected that increased maize transformation frequency can beaffected by either increased transient activity of CycE (for example,where the selectable marker, UBI::PAT-GFP::pinII, and other genes ofinterest integrate into the genome and are subsequently expressed—butwhere CycE does not integrate), or co-integration of the functional CycEexpression cassette along with the selectable marker and agronomicgene(s). Stable co-integration of CycE and PAT˜GFP is described above inthis example, and increasing transient activity is exemplified below.

Increasing Transient Activity of CycE

In order to transiently express CycE, it may be desirable to reduce thelikelihood of ectopic stable expression of the CycE gene. Strategies fortransient-only expression can be used. One such method is to express arecombinase, such as FLP, and flank the CycE expression cassette with anidentical recombinase-target-sequence, such as the FRT sequence. Underthese conditions, FLP recombinase activity will reduce stableintegration of the FRT-flanked CycE cassette, thus limiting CycEexpression to a transient interval.

Other strategies to transiently increase CycE activity include methodssuch as delivery of RNA (transcribed from the CycE gene) or CycE proteinalong with the transgene cassettes to be integrated to enhance transgeneintegration by transient stimulation of cell division. Usingwell-established methods to produce CycE-RNA, this can then be purifiedand introduced into maize cells using physical methods such asmicroinjection, bombardment, electroporation or silica fiber methods.For protein delivery, the gene is first expressed in a bacterial orbaculoviral system, the protein purified and then introduced into maizecells using physical methods such as microinjection, bombardment,electroporation or silica fiber methods.

Alternatively, CycE proteins are delivered from Argrobacteriumtumefaciens into plant cells in the form of fusions to Argrobacteriumvirulence proteins. Fusions are constructed between CycE and bacterialvirulence proteins such as VirE2, VirD2, or VirF which are known to bedelivered directly into plant cells. Fusions are constructed to retainboth those properties of bacterial virulence proteins required tomediate delivery into plant cells and the CycE activity required forenhancing transgene integration. This method should ensure a highfrequency of simultaneous co-delivery of T-DNA and functional CycEprotein into the same host cell. The methods above represent variousmeans of using the CycE gene, CycE-RNA or its encoded product toincrease transformation frequency.

Example 5 Using the CycE Gene to Improve Soybean Transformation

Delivery of the GmCycE gene can be accomplished through numerouswell-established methods for plant cells, including for example particlebombardment, sonication, PEG treatment or electroporation ofprotoplasts, electroporation of intact tissue, silica-fiber methods,microinjection or Agrobacterium-mediated transformation. Using one ofthe above methods, DNA is introduced into soybean cells capable ofgrowth on suitable soybean maize culture medium. The CycE gene (GmCycE)is cloned into a cassette with a constitutive promoter (for example, theSCP-1 promoter which confers constitutive expression in soybean, see PHIPatent application WO 99/43838) and a 3′ sequence such as the nos 3′region. Particle bombardment is used to introduce theSCP1::GmCycE::nos-containing plasmid along with aSCP1::HYG::nos-containing plasmid (which, when expressed produces aprotein which confers hygromycin resistance) into soybean cells capableof growth on suitable soybean culture medium. Such competent cells canbe from soybean suspension culture, cell culture on solid medium,freshly isolated cotyledonary nodes or meristem cells.Suspension-cultured somatic embryos of Jack, a Glycine max (I.) Merrillcultivar, are used as the target for co-delivery of a CycE and aHYG-expressing plasmid. For target tissues receiving the CycE expressioncassette, transformation frequency is improved. Media for induction ofcell cultures with high somatic embryogenic capacity, for establishingsuspensions, and for maintenance and regeneration of somatic embryos aredescribed in Bailey M A, Boerma H R, Parrott W A, 1993, Genotype effectson proliferative embryogenesis and plant regeneration of soybean, InVitro Cell Dev Biol 29P:102-108. Likewise, methods for particle-mediatedtransformation of soybean are well established in the literature, seefor example Stewart N C, Adang M J, All J N, Boerma H R, Cardineau G,Tucker D, Parrott W A, 1996, Genetic transformation, recovery andcharacterization of fertile soybean transgenic for a synthetic Bacillusthuringiensis crylAc gene, Plant Physiol 112:121-129.

Maintenance of Soybean Embryogenic Suspension Cultures

Soybean embryogenic suspension cultures are maintained in 35 ml liquidmedia SB196 or SB172 in 250 ml Erlenmeyer flasks on a rotary shaker, 150rpm, 26C with cool white fluorescent lights on 16:8 hr day/nightphotoperiod at light intensity of 30-35 uE/m2s.

Cultures are subcultured every two weeks by inoculating approximately 35mg of tissue into 35 ml of fresh liquid media. Alternatively, culturesare initiated and maintained in 6-well Costar plates.

SB 172 media is prepared as follows: (per liter), 1 bottle Murashige andSkoog Medium (Duchefa # M 0240), 1 ml B5 vitamins 1000× stock, 1 ml2,4-D stock (Gibco 11215-019), 60 g sucrose, 2 g MES, 0.667 gL-Asparagine anhydrous (GibcoBRL 11013-026), pH 5.7.

SB 196 media is prepared as follows: (per liter) 10 ml MS FeEDTA, 10 mlMS Sulfate, 10 ml FN-Lite Halides, 10 ml FN-Lite P,B,Mo, 1 ml B5vitamins 1000× stock, 1 ml 2,4-D, (Gibco 11215-019), 2.83 g KNO₃, 0.463g (NH₄)₂SO₄, 2 g MES, 1 g Asparagine Anhydrous, Powder (Gibco11013-026), 10 g Sucrose, pH 5.8.

2,4-D stock concentration 10 mg/ml is prepared as follows: 2,4-D issolubilized in 0.1 N NaOH, filter-sterilized, and stored at −20° C.

B5 vitamins 1000×stock is prepared as follows: (per 100 ml)—storealiquots at −20° C., 10 g myo-inositol, 100 mg nicotinic acid, 100 mgpyridoxine HCl, 1 g thiamine.

Particle Bombardment

Soybean embryogenic suspension cultures are transformed with variousplasmids by the method of particle gun bombardment (Klein et al., 1987;Nature, 327:70.

To prepare tissue for bombardment, approximately two flasks ofsuspension culture tissue that has had approximately 1 to 2 weeks torecover since its most recent subculture is placed in a sterile 60×20 mmpetri dish containing 1 sterile filter paper in the bottom to helpabsorb moisture. Tissue (i.e. suspension clusters approximately 3-5 mmin size) is spread evenly-across each petri plate. Residual liquid isremoved from the tissue with a pipette, or allowed to evaporate toremove excess moisture prior to bombardment. Per experiment, 4-6 platesof tissue are bombarded. Each plate is made from two flasks.

To prepare gold particles for bombardment, 30 mg gold is washed inethanol, centrifuged and resuspended in 0.5 ml of sterile water. Foreach plasmid combination (treatments) to be used for bombardment, aseparate micro-centrifuge tube is prepared, starting with 50 μl of thegold particles prepared above. Into each tube, the following are alsoadded; 5 μl of plasmid DNA (at 1 μg/μl), 50 μl CaCl₂, and 20 μl 0.1 Mspermidine. This mixture is agitated on a vortex shaker for 3 minutes,and then centrifuged using a microcentrifuge set at 14,000 RPM for 10seconds. The supernatant is decanted and the gold particles withattached, precipitated DNA are washed twice with 400 μl aliquots ofethanol (with a brief centrifugation as above between each washing). Thefinal volume of 100% ethanol per each tube is adjusted to 40 μl, andthis particle/DNA suspension is kept on ice until being used forbombardment.

Immediately before applying the particle/DNA suspension, the tube isbriefly dipped into a sonicator bath to disperse the particles, and then5 UL of DNA prep is pipetted onto each flying disk and allowed to dry.The flying disk is then placed into the Dupont Biolistics PDS1000/HE.Using the DuPont Biolistic PDS1000/HE instrument for particle-mediatedDNA delivery into soybean suspension clusters, the following settingsare used. The membrane rupture pressure is 1100 psi. The chamber isevacuated to a vacuum of 27-28 inches of mercury. The tissue is placedapproximately 3.5 inches from the retaining/stopping screen (3rd shelffrom the bottom). Each plate is bombarded twice, and the tissue clustersare rearranged using a sterile spatula between shots.

Following bombardment, the tissue is re-suspended in liquid culturemedium, each plate being divided between 2 flasks with fresh SB196 orSB172 media and cultured as described above. Four to seven dayspost-bombardment, the medium is replaced with fresh medium containing 25mg/L hygromycin (selection media). The selection media is refreshedweekly for 4 weeks and once again at 6 weeks. Weekly replacement after 4weeks may be necessary if cell density and media turbidity is high.

Four to eight weeks post-bombardment, green, transformed tissue may beobserved growing from untransformed, necrotic embryogenic clusters.Isolated, green tissue is removed and inoculated into 6-well microtiterplates with liquid medium to generate clonally-propagated, transformedembryogenic suspension cultures.

Each embryogenic cluster is placed into one well of a Costar 6-wellplate with 5 mls fresh SB196 media with 25 mg/L hygromycin. Cultures aremaintained for 2-6 weeks with fresh media changes every 2 weeks. Whenenough tissue is available, a portion of surviving transformed clonesare subcultured to a second 6-well plate as a back-up to protect againstcontamination.

In treatments where both the HYG and CycE expression cassettes aretransformed into immature embryos, a higher number of growingembryogenic cultures are expected on the selective medium and growth ofembryogenic cultures is stimulated (relative to treatments with the HYGgene alone).

Regeneration of Soybean Somatic Embryos

To promote in vitro maturation, transformed embryogenic clusters areremoved from liquid SB196 and placed on solid agar media, SB 166, for 2weeks. Tissue clumps of 2-4 mm size are plated at a tissue density of 10to 15 clusters per plate. Plates are incubated in diffuse, low light(<10 μE) at 26+/−1° C. After two weeks, clusters are subcultured to SB103 media for 3-4 weeks.

SB 166 is prepared as follows: (per liter), 1 pkg. MS salts (Gibco/BRL-Cat # 11117-017), 1 ml B5 vitamins 1000× stock, 60 g maltose, 750 mgMgCl2 hexahydrate, 5 g activated charcoal, pH 5.7, 2 g gelrite.

SB 103 media is prepared as follows: (per liter), 1 pkg. MS salts(Gibco/BRL-Cat# 11117-017), 1 ml B5 vitamins 1000× stock, 60 g maltose,750 mg MgCl2 hexahydrate, pH 5.7, 2 g gelrite.

After 5-6 week maturation, individual embryos are desiccated by placingembryos into a 100×15 petri dish with a 1 cm2 portion of the SB103 mediato create a chamber with enough humidity to promote partial desiccation,but not death.

Approximately 25 embryos are desiccated per plate. Plates are sealedwith several layers of parafilm and again are placed in a lower lightcondition. The duration of the desiccation step is best determinedempirically, and depends on size and quantity of embryos placed perplate. For example, small embryos or few embryos/plate require a shorterdrying period, while large embryos or many embryos/plate require alonger drying period. It is best to check on the embryos after about 3days, but proper desiccation will most likely take 5 to 7 days. Embryoswill decrease in size during this process.

Desiccated embryos are planted in SB 71-1 or MSO medium where they areleft to germinate under the same culture conditions described for thesuspension cultures. When the plantlets have two fully-expandedtrifoliolate leaves, germinated and rooted embryos are transferred tosterile soil and watered with MS fertilizer. Plants are grown tomaturity for seed collection and analysis. Embryogenic cultures from theCycE treatment are expected to regenerate easily. Healthy, fertiletransgenic plants are grown in the greenhouse. Seed-set on CycEtransgenic plants is expected to be similar to control plants, andtransgenic progeny are recovered.

SB 71-1 is prepared as follows: 1 bottle Gamborg's B5 salts w/sucrose(Gibco/BRL-Cat# 21153-036), 10 g sucrose, 750 mg MgCl2 hexahydrate, pH5.7, 2 g gelrite.

MSO media is prepared as follows: 1 pkg Murashige and Skoog salts (Gibco11117-066), 1 ml B5 vitamins 1000× stock, 30 g sucrose, pH 5.8, 2 gGelrite.

It is expected that higher CycE-transgene expression levels improvetransformation. For this bombardment experiment (to be performed in asimilar manner to that described above), soybean suspension cultures areused as the target tissue for bombardment. The treatments include ano-cyclin control (SCP1::HYG::nos), or the SCP1::HYG::nos marker plusone of two cyclin-expressing plasmids (SCP1::CycE::nos ornos::CycE::nos). For this experiment high levels of cyclin expression(SCP1) are compared to low levels (nos) of expression. When the SCP1promoter drives expression, the transformation frequencies for the CycEgenes are expected to be increased. Placing the CycE gene behind the nospromoter is expected to produce a transformation frequency more similarto the control. It is expected that higher expression levels result incorrespondingly higher recovery of transformants.

Example 6 Identifying Transformants in the Absence of Chemical Selection

When the CycE gene is introduced without any additional selectivemarker, transgenic calli can be identified by their ability to grow morerapidly than surrounding wild-type (non-transformed) tissues. Thisdifferential growth advantage can be used to identify CycE-transgeniccalli in the absence of conventional chemical selection (i.e. basedsolely on increased growth rates relative to the growth ofnon-transgenic callus). Transgenic callus can be verified using PCR andSouthern analysis. Northern analysis can also be used to verify whichcalli are expressing the bar gene, and which are expressing the maizeCycE gene at levels above normal wild-type cells (based on hybridizationof probes to freshly isolated mRNA population from the cells).

Inducible Expression

The CycE gene can also be cloned into a cassette with an induciblepromoter such as the benzenesulfonamide-inducible promoter. Theexpression vector is co-introduced into plant cells and after selectionon bialaphos, the transformed cells are exposed to the safener(inducer). Increased growth of CycE-transgenic callus can be observedafter the application of the safener induction. The cells are screenedfor the presence of CycE RNA by northern, or RT-PCR (using transgenespecific probes/oligo pairs), for CycE-encoded protein usingCycE-specific antibodies in Westerns or using hybridization. Cell cycleassays could also be employed, as described above.

Example 7 Control of CycE Gene Expression Using Tissue-specific orCell-specific Promoters Provides a Differential Growth Advantage

CycE gene expression using tissue-specific or cell-specific promotersstimulates cell cycle progression in the expressing tissues or cells.For example, using a seed-specific promoter will stimulate the celldivision rate and result in increased seed biomass. Alternatively,driving CycE expression with a strongly-expressed, early,tassel-specific promoter will enhance development of this entirereproductive structure.

Expression of CycE genes in other cell types and/or at different stagesof development will similarly stimulate cell division rates. Similar toresults observed in Arabidopsis (Doerner et al., 1996), root-specific orroot-preferred expression of CycE will result in larger roots and fastergrowth (i.e. more biomass accumulation).

Example 8 Meristem Transformation

Meristem transformation protocols rely on the transformation of apicalinitials or cells that can become apical initials followingreorganization due to injury or selective pressure. The progenitors ofthese apical initials differentiate to form the tissues and organs ofthe mature plant (i.e. leaves, stems, ears, tassels, etc.). Themeristems of most angiosperms are layered with each layer having its ownset of initials. Normally in the shoot apex these layers rarely mix. Inmaize the outer layer of the apical meristem, the L1, differentiates toform the epidermis while descendents of cells in the inner layer, theL2, give rise to internal plant parts including the gametes. Theinitials in each of these layers are defined solely by position and canbe replaced by adjacent cells if they are killed or compromised.Meristem transformation frequently targets a subset of the population ofapical initials and the resulting plants are chimeric. If for example, 1of 4 initials in the L1 layer of the meristem are transformed only ¼ ofepidermis would be transformed. Selective pressure can be used toenlarge sectors but this selection must be non-lethal since large groupsof cells are required for meristem function and survival. Transformationof an apical initial with a CycE expression cassette under theexpression of a promoter active in the apical meristem (either meristemspecific or constitutive) would allow the transformed cells to growfaster and displace wildtype initials driving the meristem towardshomogeneity and minimizing the chimeric nature of the plant body. Todemonstrate this, the CycE gene is cloned into a cassette with apromoter that is active within the meristem (i.e. either a strongconstitutive maize promoter such as the ubiquitin promoter including thefirst ubiquitin intron, or a promoter active in meristematic cells suchas the maize histone, cdc2 or actin promoter). Coleoptilar stage embryosare isolated and plated meristem up on a high sucrose maturation medium(see Lowe et al., 1997). The cyclin D expression cassette along with areporter construct such as Ubi:GUS:pinII can then be co-delivered(preferably 24 hours after isolation) into the exposed apical dome usingconventional particle gun transformation protocols. As a control theCycE construct can be replaced with an equivalent amount of pUC plasmidDNA. After a week to 10 days of culture on maturation medium the embryoscan be transferred to a low sucrose hormone-free germination medium.Leaves from developing plants can be sacrificed for GUS staining.Transient expression of the CycE gene in meristem cells, throughstimulation of the G1→S transition, will result in greater integrationfrequencies and hence more numerous transgenic sectors. Integration andexpression of the CycE gene will impart a competitive advantage toexpressing cells resulting in a progressive enlargement of thetransgenic sector. Due to the enhanced growth rate in CycE-expressingmeristem cells, they will supplant wild-type meristem cells as the plantcontinues to grow. The result will be both enlargement of transgenicsectors within a given cell layer (i.e. periclinal expansion) and intoadjacent cell layers (i.e. anticlinal invasions). As an increasinglylarge proportion of the meristem is occupied by CycE-expressing cells,the frequency of CycE germline inheritance should go up accordingly.

Example 9 Use of Flp/Frt System to Excise the CycE Cassette

In cases where the CycE gene has been integrated and CycE expression isuseful in the recovery of maize trangenics, but is ultimately notdesired in the final product, the CycE expression cassette (or anyportion thereof that is flanked by appropriate FRT recombinationsequences) can be excised using FLP-mediated recombination (see U.S.Pat. No. 5,929,301). In cases where transient CycE expression isdesired, FLP recombinase activity concomitant with introduction of anFRT-flanked CycE expression cassette will reduce the incidence of stableCycE integration, thus confining CycE expression and activity to atransient interval. Variations on the wild-type yeast FRT sequencehaving utility for such applications as the uses described here can befound in PHI patent application WO 09/193502.

The above examples are provided to illustrate the invention but not tolimit its scope. Other variants of the invention will be readilyapparent to one of ordinary skill in the art and are encompassed by theappended claims. All publications, patents, and patent applicationscited herein are hereby incorporated by reference.

1. An isolated ribonucleic acid sequence encoding a protein having SEQID NO:
 2. 2. An isolated nucleic acid encoding a protein having Cyclin Eactivity, wherein the nucleic acid comprises a polynucleotide thatencodes a polypeptide of SEQ ID NO:
 2. 3. An isolated nucleic acid thatmodulates the level of Cyclin E protein in a cell when compared to acorresponding cell that does not contain the isolated nucleic acid,wherein the nucleic acid comprises a polynucleotide having the sequenceset forth in SEQ ID NO:
 1. 4. An isolated nucleic acid that modulatesthe level of Cyclin E protein in a cell when compared to the level ofCyclin E in a corresponding cell that does not contain the isolatednucleic acid, wherein the nucleic acid comprises a polynuoleotide fullycomplementary to the sequence set forth in SEQ ID NO: 1.